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Sample records for actin layer line

  1. Intensity increases of actin layer-lines on activation of the Limulus muscle.

    PubMed Central

    Maéda, Y; Boulin, C; Gabriel, A; Sumner, I; Koch, M H

    1986-01-01

    Small angle x-ray diffraction patterns were recorded from isometrically contracting Limulus (horseshoe crab) telson levator muscle using a multiwire proportional-area detector on the storage ring DORIS. In the pattern a substantial increase in intensity is observed on the thin-filament-associated layer-line at 1/38 nm-1 (the first actin layer-line) with a maximum increase at a radial spacing of R = 0.07 nm-1 but there is a much smaller change in the intensity of the 5.9-nm layer-line, which also arises from the thin filament structure. The results suggest that during contraction the myosin heads, presumably being attached to the thin filaments, are arranged along the long-stranded helical tracks of the thin filaments but that the spatial relationship between the heads and the actin monomers varies. Intensity increases have also been observed (Maéda et al., manuscript in preparation) in the part of the patterns from frog muscle and barnacle muscle, which are attributable to the first actin layer-line. It is thus likely that the intensity increase of the first actin layer-line on the Limulus pattern is associated not with structural features which are special to Limulus muscle, but with the tension generating processes that are shared by muscles in general. Images FIGURE 1 FIGURE 2 PMID:3801566

  2. Actin marker lines in grapevine reveal a gatekeeper function of guard cells.

    PubMed

    Guan, Xin; Buchholz, Günther; Nick, Peter

    2014-08-15

    Resistance to abiotic and biotic stress is a central topic for sustainable agriculture, especially in grapevine, one of the field crops with the highest economic output per acreage. As early cellular factors for plant defense, actin microfilaments (AF) are of high relevance. We therefore generated a transgenic actin marker line for grapevine by expressing a fusion protein between green fluorescent protein and the second actin-binding domain of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. Based on this first cytoskeletal-marker line in grapevine, the response of AFs to phytopathogenic microorganisms could be followed in vivo. Upon inoculation with fluorescently labeled strains of phytopathogenic bacteria, actin responses were confined to the guard cells. In contrast, upon contact with zoospores of Plasmopara viticola, not only the guard cells, but also epidermal pavement cells, where no zoospores had attached responded with the formation of a perinuclear actin basket. Our data support the hypothesis that guard cells act as pacemakers of defense, dominating the responses of the remaining epidermal cells. PMID:24973589

  3. Actin marker lines in grapevine reveal a gatekeeper function of guard cells.

    PubMed

    Guan, Xin; Buchholz, Günther; Nick, Peter

    2014-08-15

    Resistance to abiotic and biotic stress is a central topic for sustainable agriculture, especially in grapevine, one of the field crops with the highest economic output per acreage. As early cellular factors for plant defense, actin microfilaments (AF) are of high relevance. We therefore generated a transgenic actin marker line for grapevine by expressing a fusion protein between green fluorescent protein and the second actin-binding domain of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. Based on this first cytoskeletal-marker line in grapevine, the response of AFs to phytopathogenic microorganisms could be followed in vivo. Upon inoculation with fluorescently labeled strains of phytopathogenic bacteria, actin responses were confined to the guard cells. In contrast, upon contact with zoospores of Plasmopara viticola, not only the guard cells, but also epidermal pavement cells, where no zoospores had attached responded with the formation of a perinuclear actin basket. Our data support the hypothesis that guard cells act as pacemakers of defense, dominating the responses of the remaining epidermal cells.

  4. Double layers on auroral field lines

    NASA Technical Reports Server (NTRS)

    Hudson, M. K.; Lotko, W.; Witt, E.

    1982-01-01

    Time-stationary solutions to the Vlasov-Poisson equation for ion holes and double layers were examined along with particle simulations which pertain to recent observations of small amplitude (e phi)/t sub e approx. 1 electric field structures on auroral field lines. Both the time-stationary analysis and the simulations suggest that double layers evolve from holes in ion phase space when their amplitude reaches (e phi)/t sub e approx. 1. Multiple small amplitude double layers which are seen in long simulation systems and are seen to propagate past spacecraft may account for the acceleration of plasma sheet electrons to produce the discrete aurora.

  5. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

    PubMed

    Fujiwara, Takahiro K; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A; Watanabe, Yusuke; Umemura, Yasuhiro M; Murakoshi, Hideji; Suzuki, Kenichi G N; Nemoto, Yuri L; Morone, Nobuhiro; Kusumi, Akihiro

    2016-04-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed "hop diffusion") for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion.

  6. Reinforcing the LINC complex connection to actin filaments: the role of FHOD1 in TAN line formation and nuclear movement

    PubMed Central

    Antoku, Susumu; Zhu, Ruijun; Kutscheidt, Stefan; Fackler, Oliver T; Gundersen, Gregg G

    2015-01-01

    Positioning the nucleus is critical for many cellular processes including cell division, migration and differentiation. The linker of nucleoskeleton and cytoskeleton (LINC) complex spans the inner and outer nuclear membranes and has emerged as a major factor in connecting the nucleus to the cytoskeleton for movement and positioning. Recently, we discovered that the diaphanous formin family member FHOD1 interacts with the LINC complex component nesprin-2 giant (nesprin-2G) and that this interaction plays essential roles in the formation of transmembrane actin-dependent nuclear (TAN) lines and nuclear movement during cell polarization in fibroblasts. We found that FHOD1 strengthens the connection between nesprin-2G and rearward moving dorsal actin cables by providing a second site of interaction between nesprin-2G and the actin cable. These results indicate that the LINC complex connection to the actin cytoskeleton can be enhanced by cytoplasmic factors and suggest a new model for TAN line formation. We discuss how the nesprin-2G-FHOD1 interaction may be regulated and its possible functional significance for development and disease. PMID:26083340

  7. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane

    PubMed Central

    Fujiwara, Takahiro K.; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A.; Watanabe, Yusuke; Umemura, Yasuhiro M.; Murakoshi, Hideji; Suzuki, Kenichi G. N.; Nemoto, Yuri L.; Morone, Nobuhiro; Kusumi, Akihiro

    2016-01-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed “hop diffusion”) for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion. PMID:26864625

  8. Actinic Keratoses

    PubMed Central

    Brown, Marc D.

    2009-01-01

    Actinic keratoses are common intra-epidermal neoplasms that lie on a continuum with squamous cell carcinoma. Tightly linked to ultraviolet irradiation, they occur in areas of chronic sun exposure, and early treatment of these lesions may prevent their progression to invasive disease. A large variety of effective treatment modalities exist, and the optimal therapeutic choice is dependent on a variety of patient- and physician-associated variables. Many established and more recent approaches are discussed in this review with a focus on efficacy and administration techniques. Several previously experimental options, such as imiquimod and photodynamic therapy, have become incorporated as first-line options for the treatment of actinic keratoses, while combination treatment strategies have been gaining in popularity. The goal of all therapies is to ultimately limit the morbidity and mortality of squamous cell carcinoma. (J Clin Aesthetic Dermatol. 2009;2(7):43–48.) PMID:20729970

  9. Actinic Keratosis

    MedlinePlus

    ... rashes clinical tools newsletter | contact Share | Actinic Keratosis (Solar Keratosis) Information for adults A A A Actinic ... the touch. Overview Actinic keratoses, also known as solar keratoses, are small rough or scaly areas of ...

  10. Actin in Herpesvirus Infection

    PubMed Central

    Roberts, Kari L.; Baines, Joel D.

    2011-01-01

    Actin is important for a variety of cellular processes, including uptake of extracellular material and intracellular transport. Several emerging lines of evidence indicate that herpesviruses exploit actin and actin-associated myosin motors for viral entry, intranuclear transport of capsids, and virion egress. The goal of this review is to explore these processes and to highlight potential future directions for this area of research. PMID:21994736

  11. Actin dynamics in living mammalian cells.

    PubMed

    Ballestrem, C; Wehrle-Haller, B; Imhof, B A

    1998-06-01

    The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human &bgr ;-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured 'actin clouds' were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

  12. Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status

    SciTech Connect

    Djuzenova, Cholpon S.; Fiedler, Vanessa; Memmel, Simon; Katzer, Astrid; Hartmann, Susanne; Krohne, Georg; Zimmermann, Heiko; Polat, Bülent; Flentje, Michael; and others

    2015-01-15

    Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion. - Highlights: • We examine 5 glioblastoma lines on the invasion capacity and actin cytoskeleton. • Glioblastoma cell lines mutated in both p53 and PTEN were the most invasive. • Less invasive cells showed much less lamellipodia, but more actin stress fibers. • A mechanism for the differences in tumor cell invasion is proposed.

  13. A supracellular system of actin-lined canals controls biogenesis and release of virulence factors in parasitoid venom glands.

    PubMed

    Ferrarese, Roberto; Morales, Jorge; Fimiarz, Daniel; Webb, Bruce A; Govind, Shubha

    2009-07-01

    Parasitoid wasps produce virulence factors that bear significant resemblance to viruses and have the ability to block host defense responses. The function of these virulence factors, produced predominantly in wasp venom glands, and the ways in which they interfere with host development and physiology remain mysterious. Here, we report the discovery of a specialized system of canals in venom glands of five parasitoid wasps that differ in their infection strategies. This supracellular canal system is made up of individual secretory units, one per secretory cell. Individual units merge into the canal lumen. The membrane surface of the proximal end of each canal within the secretory cell assumes brush border morphology, lined with bundles of F-actin. Systemic administration of cytochalasin D compromises the integrity of the secretory unit. We show a dynamic and continuous association of p40, a protein of virus-like particles from a Drosophila parasitoid, L. heterotoma, with the canal and venom gland lumen. Similar structures in three Leptopilina species and Ganaspis xanthopoda, parasitoids of Drosophila spp., and Campoletis sonorenesis, a parasitoid of Heliothis virescens, suggest that this novel supracellular canal system is likely to be a common trait of parasitoid venom glands that is essential for efficient biogenesis and delivery of virulence factors. PMID:19561216

  14. A supracellular system of actin-lined canals controls biogenesis and release of virulence factors in parasitoid venom glands

    PubMed Central

    Ferrarese, Roberto; Morales, Jorge; Fimiarz, Daniel; Webb, Bruce A.; Govind, Shubha

    2009-01-01

    Summary Parasitoid wasps produce virulence factors that bear significant resemblance to viruses and have the ability to block host defense responses. The function of these virulence factors, produced predominantly in wasp venom glands, and the ways in which they interfere with host development and physiology remain mysterious. Here, we report the discovery of a specialized system of canals in venom glands of five parasitoid wasps that differ in their infection strategies. This supracellular canal system is made up of individual secretory units, one per secretory cell. Individual units merge into the canal lumen. The membrane surface of the proximal end of each canal within the secretory cell assumes brush border morphology, lined with bundles of F-actin. Systemic administration of cytochalasin D compromises the integrity of the secretory unit. We show a dynamic and continuous association of p40, a protein of virus-like particles from a Drosophila parasitoid, L. heterotoma, with the canal and venom gland lumen. Similar structures in three Leptopilina species and Ganaspis xanthopoda, parasitoids of Drosophila spp., and Campoletis sonorenesis, a parasitoid of Heliothis virescens, suggest that this novel supracellular canal system is likely to be a common trait of parasitoid venom glands that is essential for efficient biogenesis and delivery of virulence factors. PMID:19561216

  15. The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

    PubMed Central

    Stauffer, Sarah; Rahman, Sheikh Abdul; de Marco, Alex; Carlson, Lars-Anders; Glass, Bärbel; Oberwinkler, Heike; Herold, Nikolas; Briggs, John A. G.; Müller, Barbara

    2014-01-01

    ABSTRACT Actin and actin-binding proteins are incorporated into HIV-1 particles, and F-actin has been suggested to bind the NC domain in HIV-1 Gag. Furthermore, F-actin has been frequently observed in the vicinity of HIV-1 budding sites by cryo-electron tomography (cET). Filamentous structures emanating from viral buds and suggested to correspond to actin filaments have been observed by atomic force microscopy. To determine whether the NC domain of Gag is required for actin association with viral buds and for actin incorporation into HIV-1, we performed comparative analyses of virus-like particles (VLPs) obtained by expression of wild-type HIV-1 Gag or a Gag variant where the entire NC domain had been replaced by a dimerizing leucine zipper [Gag(LZ)]. The latter protein yielded efficient production of VLPs with near-wild-type assembly kinetics and size and exhibited a regular immature Gag lattice. Typical HIV-1 budding sites were detected by using cET in cells expressing either Gag or Gag(LZ), and no difference was observed regarding the association of buds with the F-actin network. Furthermore, actin was equally incorporated into wild-type HIV-1 and Gag- or Gag(LZ)-derived VLPs, with less actin per particle observed than had been reported previously. Incorporation appeared to correlate with the relative intracellular actin concentration, suggesting an uptake of cytosol rather than a specific recruitment of actin. Thus, the NC domain in HIV-1 Gag does not appear to have a role in actin recruitment or actin incorporation into HIV-1 particles. IMPORTANCE HIV-1 particles bud from the plasma membrane, which is lined by a network of actin filaments. Actin was found to interact with the nucleocapsid domain of the viral structural protein Gag and is incorporated in significant amounts into HIV-1 particles, suggesting that it may play an active role in virus release. Using electron microscopy techniques, we previously observed bundles of actin filaments near HIV-1 buds

  16. Actin stress in cell reprogramming

    PubMed Central

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

    2014-01-01

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

  17. Development of a cell line from Echinococcus granulosus germinal layer.

    PubMed

    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  18. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar) ... Actinic keratosis is caused by exposure to sunlight. You are more likely to develop it if you: Have fair skin, blue or green eyes, or blond or red hair Had a ...

  19. Dynamics of shear velocity layer with bent magnetic field lines

    NASA Astrophysics Data System (ADS)

    Galinsky, V. L.; Sonnerup, B. U. Ö.

    A fully three-dimensional, magnetohydro-dynamic simulation of velocity-sheared plasma flow in an ambient transverse magnetic field with bent magnetic field lines has been performed. “Ionospheric-like” boundary conditions were used for closing field-aligned currents, the two ionospheres being represented by conducting plates with constant resistivity. Compared to the standard plane 2D case with a uniform transverse magnetic field, the growth rate of the Kelvin-Helmholtz instability drops significantly as bending increases. Under conditions representative of the Earth's low latitude boundary layer, the instability may be suppressed completely by the magnetic field-line tension if the field-line bending is sufficiently strong. For weak bending, a combination of the tearing mode instability and the Kelvin-Helmholtz instability leads to the formation of localized 3D current/vortex tubes, the ionospheric foot prints of which are possible models of the auroral bright spots observed by the Viking satellite.

  20. Actinic Cheilitis

    MedlinePlus

    ... is a precancerous condition related to cumulative lifetime sun exposure. The lower lip is most often affected. Individuals ... Wearing barrier clothing (eg, wide-brimmed hats) and sunscreen-containing lip balms can aid in preventing actinic ...

  1. Radial transmission line analysis of multi-layer structures

    SciTech Connect

    Hahn, H.; Hammons, L.

    2011-03-28

    The analysis of multi-layer beam tubes is a standard problem and involves axially propagating waves. This treatment is ill suited to a short multi-layer structure such as the present example of a ferrite covered ceramic break in the beam tube at the ERL photo-cathode electron gun. This paper demonstrates that such structure can better be treated by radial wave propagation. The theoretical method is presented and numerical results are compared with measured network analyser data and Microwave Studio generated simulations. The results confirm the concept of radial transmission lines as a valid analytical method. An Energy Recovery Linac (ERL) is being constructed at this laboratory for the purpose of research towards an envisioned Electron Ion Collider. One of the pertinent topics is damping of Higher Order Modes (HOM). In this ERL, the damping is provided by ferrite absorbers in the beam tube. A modified version thereof, a ceramic break surrounded by ferrite, is planed for the superconducting electron gun. The damper here is located at room temperature just outside of the gun. If used in a cavity chain, the ceramic break is in the vacuum tube at helium temperature whereas the ferrite is moved into the cryostat insulating vacuum allowing higher temperatures. The general properties of the ferrite HOM dampers have been published but are more detailed in this paper.

  2. Dynamic actin structures stabilized by profilin.

    PubMed Central

    Finkel, T; Theriot, J A; Dise, K R; Tomaselli, G F; Goldschmidt-Clermont, P J

    1994-01-01

    We describe the production and analysis of clonal cell lines in which we have overexpressed human profilin, a small ubiquitous actin monomer binding protein, to assess the role of profilin on actin function in vivo. The concentration of filamentous actin is increased in cells with higher profilin levels, and actin filament half-life measured in these cells is directly proportional to the steady-state profilin concentration. The distribution of actin filaments is altered by profilin overexpression. While parallel actin bundles crossing the cells are virtually absent in cells overexpressing profilin, the submembranous actin network of these cells is denser than in control cells. These results suggest that in vivo profilin regulates the stability, and thereby distribution, of specific dynamic actin structures. Images PMID:8108438

  3. An α-smooth muscle actin (acta2/αsma) zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells.

    PubMed

    Whitesell, Thomas R; Kennedy, Regan M; Carter, Alyson D; Rollins, Evvi-Lynn; Georgijevic, Sonja; Santoro, Massimo M; Childs, Sarah J

    2014-01-01

    Mural cells of the vascular system include vascular smooth muscle cells (SMCs) and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of mural cell development in vertebrates is α smooth muscle actin (acta2; αsma), which is expressed by pericytes and SMCs. In vivo models of vascular mural cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing cells. These transgenic fish were used to trace the live development of mural cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive cells and the number of types of blood vessels associated with mural cells increases. Interestingly, the mural cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish mural cells develop relatively late, and have little mobility once they associate with vessels.

  4. [Actinic Keratosis].

    PubMed

    Dejaco, D; Hauser, U; Zelger, B; Riechelmann, H

    2015-07-01

    Actinic keratosis is a cutaneous lesion characterized by proliferation of atypical epidermal keratinocytes due to prolonged exposure to exogenous factors such as ultraviolet radiation. AKs are in-situ-squamous cell carcinomas (PEC) of the skin. AK typically presents as erythematous, scaly patch or papule (classic AK), occasionally as thick, adherent scale on an erythematous base. Mostly fair-skinned adults are affected. AKs typically occur in areas of frequent sun exposure (balding scalp, face, "H-region", lateral neck, décolleté, dorsum of the hand and lower extremities). Actinic Cheilitis is the term used for AKs appearing on the lips. The diagnosis of AK is based on clinical examination including inspection and palpation. The typical palpable rough surface of AK often precedes a visible lesion. Dermoscopy may provide additional information. If diagnosis is uncertain and invasion suspected, biopsy and histopathologic evaluation should be performed. The potential for progression to invasive PECs mandates therapeutic intervention. Treatment options include topical and systemic therapies. Topical therapies are classified into physical, medical and combined physical-chemical approaches and a sequential combination of treatment modalities is possible. Topical-physical cryotherapy is the treatment of choice for isolated, non-hypertrophic AK. Topical-medical treatment, e. g. 5-fluoruracil (5FU) cream or Imiquomod or Ingenolmebutat application is used for multiple, non-hypertrophic AKs. For hypertrophic AKs, a dehorning pretreatment with salicinated vaseline is recommended. Isolated hypertrophic AKs often need cryotherapy with prolonged freezing time or several consecutive applications. Sequentially combined approaches are recommended for multiple, hypertrophic AKs. Photodynamic therapy (PDT) as example for a combined physical-chemical approach is an established treatment for multiple, non-hypertrophic and hypertrophic AKs. Prevention includes avoidance of sun and

  5. Skeletal and cardiac α-actin isoforms differently modulate myosin cross-bridge formation and myofibre force production.

    PubMed

    Ochala, Julien; Iwamoto, Hiroyuki; Ravenscroft, Gianina; Laing, Nigel G; Nowak, Kristen J

    2013-11-01

    Multiple congenital myopathies, including nemaline myopathy, can arise due to mutations in the ACTA1 gene encoding skeletal muscle α-actin. The main characteristics of ACTA1 null mutations (absence of skeletal muscle α-actin) are generalized skeletal muscle weakness and premature death. A mouse model (ACTC(Co)/KO) mimicking these conditions has successfully been rescued by transgenic over-expression of cardiac α-actin in skeletal muscles using the ACTC gene. Nevertheless, myofibres from ACTC(Co)/KO animals generate less force than normal myofibres (-20 to 25%). To understand the underlying mechanisms, here we have undertaken a detailed functional study of myofibres from ACTC(Co)/KO rodents. Mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized myofibres showed, upon maximal Ca(2+) activation and under rigor conditions, lower stiffness and disrupted actin-layer line reflections in ACTC(Co)/KO when compared with age-matched wild-types. These results demonstrate that in ACTC(Co)/KO myofibres, the presence of cardiac α-actin instead of skeletal muscle α-actin alters actin conformational changes upon activation. This later finely modulates the strain of individual actomyosin interactions and overall lowers myofibre force production. Taken together, the present findings provide novel primordial information about actin isoforms, their functional differences and have to be considered when designing gene therapies for ACTA1-based congenital myopathies. PMID:23784376

  6. Actin filament organization and myosin head labelling patterns in vertebrate skeletal muscles in the rigor and weak binding states.

    PubMed

    Squire, J M; Harford, J J

    1988-08-01

    The structures of vertebrate skeletal muscles (particularly from frog and fish) in the rigor state are analysed in terms of the concept of target areas on actin filaments. Assuming that 100% of the heads are to be attached to actin in rigor, then satisfactory qualitative low-resolution modelling of observed X-ray diffraction data is obtained if the outer ends of these myosin heads can move axially (total range about 200A) and azimuthally (total range less than 60 degrees) from their original lattice sites on the myosin filament surface to attach in defined target areas on the actin filaments. On this basis, each actin target area comprises about four actin monomers along one of the two long-pitched helical strands of the actin filament (about 200 A) or an azimuthal range of actin binding sites of about 100 degrees around the thin filament axis. If myosin heads simply label in a non-specific way the nearest actin monomers to them, as could occur with non-specific transient attachment in a 'weak binding' state, then the predicted X-ray diffraction pattern would comprise layer lines at the same axial spacings (orders of 429 A) as those seen in patterns from resting muscle. It is shown that actin target areas in vertebrate skeletal muscles are probably arranged on an approximate 62 (right-handed) helix of pitch (P) of about 720 A, subunit translation P/6 and near repeat P/2. Troponin position need not be considered in defining the labelling pattern of cross-bridges on this 62 helix of target areas; the target areas appear to be defined solely by the azimuthal position of the actin binding sites. The distribution of actin filament labelling patterns could be regular in fish muscle which has a 'crystalline' A-band, but will be irregular in higher vertebrate muscles such as frog sartorius muscle.

  7. Human epidermal growth factor for the stratification of synovial lining layer and neovascularisation in rheumatoid arthritis.

    PubMed Central

    Shiozawa, S; Shiozawa, K; Tanaka, Y; Morimoto, I; Uchihashi, M; Fujita, T; Hirohata, K; Hirata, Y; Imura, S

    1989-01-01

    Immunohistochemical study showed selective localisation of human epidermal growth factor (hEGF) to the synovial lining layer. Although the synovial lining layer of the rheumatoid, osteoarthritic, and traumatic joints was hEGF positive, hEGF staining was especially dense at the rheumatoid synovial lining layer; the staining increasing linearly according to the degree of stratification of the lining layer (r = 1). Human epidermal growth factor was ultrastructurally localised to cytoplasm, especially to rough endoplasmic reticulum, of the synovial lining fibroblast-like (type B) cell. Only the cell surface of macrophage-like (type A) cells was hEGF positive. When different histological variables were compared with each other a positive correlation was found between hEGF staining of the synovial lining layer and the degree of neovascularisation of rheumatoid synovium (r = 0.72). Although some lymphocytes were weakly hEGF positive, neovascularisation did not correlate with the extent of lymphocyte infiltration or of hEGF staining of lymphocytes. Lymphocyte infiltration or hEGF staining of lymphocytes did not correlate with hEGF staining of the synovial lining layer, whereas the lymphocyte infiltration correlated positively with the extent of perivascular accumulation of lymphocytes (r = 0.89). These findings suggest that (a) hEGF is synthesised by and secreted through endoplasmic reticulum and Golgi apparatus from the synovial lining type B cell; (b) hEGF is at least partially responsible for the pathogenesis of stratification of the rheumatoid synovial lining layer, and perhaps of neovascularisation of the rheumatoid synovium, whereas it is not responsible for lymphocyte accumulation to the rheumatoid synovium. Images PMID:2479344

  8. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  9. On motion of fluid in boundary layer near line of intersection of two planes

    NASA Technical Reports Server (NTRS)

    Loitsianskii, L G; Bolshakov, V P

    1951-01-01

    In the paper "The Mutual Interference of Boundary Layers," the authors investigated the problem of the interference of two planes intersecting at right angles on the boundary layers formed by the motion of fluid along the line of intersection of these planes. In the present paper, the results of the preceding one are generalized to the case of planes intersecting at any angle. The motion of a fluid in an angle less than 180 degrees is discussed and the enlargement of the boundary layers near the line of intersection of the planes, the limits of the interference effects of the boundary layers, and the corrections on the drag are determined. All computations are conducted by the Karman-Pohlhausen method for laminar and turbulent boundary layers. The results are reduced to tabulated form.

  10. Predicting Anticancer Drug Responses Using a Dual-Layer Integrated Cell Line-Drug Network Model

    PubMed Central

    Fang, Yun; Wang, Jun; Zheng, Xiaoqi; Liu, X. Shirley

    2015-01-01

    The ability to predict the response of a cancer patient to a therapeutic agent is a major goal in modern oncology that should ultimately lead to personalized treatment. Existing approaches to predicting drug sensitivity rely primarily on profiling of cancer cell line panels that have been treated with different drugs and selecting genomic or functional genomic features to regress or classify the drug response. Here, we propose a dual-layer integrated cell line-drug network model, which uses both cell line similarity network (CSN) data and drug similarity network (DSN) data to predict the drug response of a given cell line using a weighted model. Using the Cancer Cell Line Encyclopedia (CCLE) and Cancer Genome Project (CGP) studies as benchmark datasets, our single-layer model with CSN or DSN and only a single parameter achieved a prediction performance comparable to the previously generated elastic net model. When using the dual-layer model integrating both CSN and DSN, our predicted response reached a 0.6 Pearson correlation coefficient with observed responses for most drugs, which is significantly better than the previous results using the elastic net model. We have also applied the dual-layer cell line-drug integrated network model to fill in the missing drug response values in the CGP dataset. Even though the dual-layer integrated cell line-drug network model does not specifically model mutation information, it correctly predicted that BRAF mutant cell lines would be more sensitive than BRAF wild-type cell lines to three MEK1/2 inhibitors tested. PMID:26418249

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

    PubMed

    Brugerolle, G; Bricheux, G; Coffe, G

    1996-01-01

    The flagellate form of Trichomonas vaginalis (T v) transforms to amoeboid cells upon adherence to converslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T v F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T v adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum. Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a 'trichomonas branch' and a 'tritrichomonas branch' is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads. PMID:9175265

  12. Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

    PubMed Central

    Cunningham, C. Casey; Leclerc, Nicole; Flanagan, Lisa A.; Lu, Mei; Janmey, Paul A.; Kosik, Kenneth S.

    1997-01-01

    The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments. PMID:9049250

  13. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    SciTech Connect

    Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K.

    2008-09-03

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

  14. Coupling Between Microstrip Lines Embedded in Polyimide Layers for 3D-MMICs on Si

    NASA Technical Reports Server (NTRS)

    Ponchak, George E.; Tentzeris, Emmanouil M.; Papapolymerou, John

    2001-01-01

    Three-dimensional circuits built upon multiple layers of polyimide are required for constructing Si/SiGe monolithic microwave/millimeter-wave integrated circuits on CMOS (low resistivity) Si wafers. However, the closely spaced transmission lines are susceptible to high levels of coupling, which degrades circuit performance. In this paper, Finite Difference Time Domain (FDTD) analysis and measured characteristics of novel shielding structures that significantly reduce coupling between embedded microstrip lines are presented.

  15. Position and Orientation of Phalloidin in F-Actin Determined by X-Ray Fiber Diffraction Analysis

    PubMed Central

    Oda, Toshiro; Namba, Keiichi; Maéda, Yuichiro

    2005-01-01

    Knowledge of the phalloidin binding position in F-actin and the relevant understanding of the mechanism of F-actin stabilization would help to define the structural characteristics of the F-actin filament. To determine the position of bound phalloidin experimentally, x-ray fiber diffraction data were obtained from well-oriented sols of F-actin and the phalloidin-F-actin complex. The differences in the layer-line intensity distributions, which were clearly observed even at low resolution (8 Å), produced well-resolved peaks corresponding to interphalloidin vectors in the cylindrically averaged difference-Patterson map, from which the radial binding position was determined to be ∼10 Å from the filament axis. Then, the azimuthal and axial positions were determined by single isomorphous replacement phasing and a cross-Patterson map in radial projection to be ∼84° and 0.5 Å relative to the actin mass center. The refined position was close to the position found by prior researchers. The position of rhodamine attached to phalloidin in the rhodamine-phalloidin-F-actin complex was also determined, in which the conjugated Leu(OH)7 residue was found to face the outside of the filament. The position and orientation of the bound phalloidin so determined explain the increase in the interactions between long-pitch strands of F-actin and would also account for the inhibition of phosphate release, which might also contribute to the F-actin stabilization. The method of analysis developed in this study is applicable for the determination of binding positions of other drugs, such as jasplakinolide and dolastatin 11. PMID:15653738

  16. Remarkably low-energy one-dimensional fault line defects in single-layered phosphorene.

    PubMed

    Jang, Woosun; Kang, Kisung; Soon, Aloysius

    2015-12-01

    Systematic engineering of atomic-scale low-dimensional defects in two-dimensional nanomaterials is a promising method to modulate the electronic properties of these nanomaterials. Defects at interfaces such as grain boundaries and line defects can often be detrimental to technologically important nanodevice operations and thus a fundamental understanding of how such one-dimensional defects may have an influence on their physio-chemical properties is pivotal for optimizing their device performance. Of late, two-dimensional phosphorene has attracted much attention due to its high carrier mobility and good mechanical flexibility. In this study, using density-functional theory, we have investigated the temperature-dependent energetics and electronic structure of single-layered phosphorene with various fault line defects. We have generated different line defect models based on a fault method, rather than the conventional rotation method. This has allowed us to study and identify new low-energy line defects, and we show how these low-energy line defects could well modulate the electronic band gap energies of single-layered two-dimensional phosphorene - offering a range of metallic to semiconducting properties in these newly proposed low-energy line defects in phosphorene.

  17. Actin Rings of Power.

    PubMed

    Schwayer, Cornelia; Sikora, Mateusz; Slováková, Jana; Kardos, Roland; Heisenberg, Carl-Philipp

    2016-06-20

    Circular or ring-like actin structures play important roles in various developmental and physiological processes. Commonly, these rings are composed of actin filaments and myosin motors (actomyosin) that, upon activation, trigger ring constriction. Actomyosin ring constriction, in turn, has been implicated in key cellular processes ranging from cytokinesis to wound closure. Non-constricting actin ring-like structures also form at cell-cell contacts, where they exert a stabilizing function. Here, we review recent studies on the formation and function of actin ring-like structures in various morphogenetic processes, shedding light on how those different rings have been adapted to fulfill their specific roles. PMID:27326928

  18. Nematic textures in F-actin

    NASA Astrophysics Data System (ADS)

    Das, P.; Roy, J.; Chakrabarti, N.; Basu, S.; Das, U.

    2002-05-01

    Actin filaments, which are protein polymers occurring abundantly and ubiquitously in muscle and nonmuscle cells, are known to align in a shear flow, and with an external magnetic field. They form a nematic liquid crystal of the athermal type at a low concentration. Typical defects and textures of the nematic actin liquid crystal are described in this work. The generation of well-aligned nematic single crystals has been reported, in the vicinity of an air-water interface, with the actin filaments spontaneously aligning normal to the interface. Away from the air-water interface nematic single crystal domains are due to the alignment of the actin filaments parallel to the glass surface. The twist-bend nature of the disclination line of integral strength (m=1) has been attributed to the relative magnitudes of the anisotropic curvature elastic constants, which reflect the filaments' semirigidity.

  19. A moving contact line as a rheometer for nanometric interfacial layers

    PubMed Central

    Lhermerout, Romain; Perrin, Hugo; Rolley, Etienne; Andreotti, Bruno; Davitt, Kristina

    2016-01-01

    How a liquid drop sits or moves depends on the physical and mechanical properties of the underlying substrate. This can be seen in the hysteresis of the contact angle made by a drop on a solid, which is known to originate from surface heterogeneities, and in the slowing of droplet motion on deformable solids. Here, we show how a moving contact line can be used to characterize a molecularly thin polymer layer on a solid. We find that the hysteresis depends on the polymerization index and can be optimized to be vanishingly small (<0.07°). The mechanical properties are quantitatively deduced from the microscopic contact angle, which is proportional to the speed of the contact line and the Rouse relaxation time divided by the layer thickness, in agreement with theory. Our work opens the prospect of measuring the properties of functionalized interfaces in microfluidic and biomedical applications that are otherwise inaccessible. PMID:27562022

  20. A moving contact line as a rheometer for nanometric interfacial layers.

    PubMed

    Lhermerout, Romain; Perrin, Hugo; Rolley, Etienne; Andreotti, Bruno; Davitt, Kristina

    2016-08-26

    How a liquid drop sits or moves depends on the physical and mechanical properties of the underlying substrate. This can be seen in the hysteresis of the contact angle made by a drop on a solid, which is known to originate from surface heterogeneities, and in the slowing of droplet motion on deformable solids. Here, we show how a moving contact line can be used to characterize a molecularly thin polymer layer on a solid. We find that the hysteresis depends on the polymerization index and can be optimized to be vanishingly small (<0.07°). The mechanical properties are quantitatively deduced from the microscopic contact angle, which is proportional to the speed of the contact line and the Rouse relaxation time divided by the layer thickness, in agreement with theory. Our work opens the prospect of measuring the properties of functionalized interfaces in microfluidic and biomedical applications that are otherwise inaccessible.

  1. A moving contact line as a rheometer for nanometric interfacial layers

    NASA Astrophysics Data System (ADS)

    Lhermerout, Romain; Perrin, Hugo; Rolley, Etienne; Andreotti, Bruno; Davitt, Kristina

    2016-08-01

    How a liquid drop sits or moves depends on the physical and mechanical properties of the underlying substrate. This can be seen in the hysteresis of the contact angle made by a drop on a solid, which is known to originate from surface heterogeneities, and in the slowing of droplet motion on deformable solids. Here, we show how a moving contact line can be used to characterize a molecularly thin polymer layer on a solid. We find that the hysteresis depends on the polymerization index and can be optimized to be vanishingly small (<0.07°). The mechanical properties are quantitatively deduced from the microscopic contact angle, which is proportional to the speed of the contact line and the Rouse relaxation time divided by the layer thickness, in agreement with theory. Our work opens the prospect of measuring the properties of functionalized interfaces in microfluidic and biomedical applications that are otherwise inaccessible.

  2. Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin.

    PubMed

    Kensler, Robert W; Shaffer, Justin F; Harris, Samantha P

    2011-04-01

    Cardiac myosin-binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin-binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2 (5-30μM, in a buffer containing in mM: 180 KCl, 1 MgCl(2), 1 EDTA, 1 DTT, 20 imidazole, at pH 7.4) with F-actin (5μM) for 30min and examined negatively-stained samples of the solution by electron microscopy (EM). Examination of EM images revealed that C0-C2 bound to F-actin to form long helically-ordered complexes. Fourier transforms indicated that C0-C2 binds with the helical periodicity of actin with strong 1st and 6th layer lines. The results provide direct evidence that the N-terminus of cMyBP-C can bind to F-actin in a periodic complex. This interaction of cMyBP-C with F-actin supports the possibility that binding of cMyBP-C to F-actin may play a role in the regulation of cardiac contraction.

  3. Direct dynamin–actin interactions regulate the actin cytoskeleton

    PubMed Central

    Gu, Changkyu; Yaddanapudi, Suma; Weins, Astrid; Osborn, Teresia; Reiser, Jochen; Pollak, Martin; Hartwig, John; Sever, Sanja

    2010-01-01

    The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs. PMID:20935625

  4. Initiation of deep convection along boundary layer convergence lines in a semitropical environment

    SciTech Connect

    Fankhauser, J.C.; Crook, N.A.; Tuttle, J.; Miller, L.J.; Wade, C.G.

    1995-02-01

    The initiation of deep convection through forcing along boundary layer convergence lines is examined using observations from the Convection and Precipitation/Electrification (CaPE) Experiment conducted in east-central Florida during the summer of 1991. The study is concerned with the evolution and interaction of two converging air masses that were initially separated by an intervening boundary layer characterized by neutral stability and horizontal convective rolls. As anticipated, major thunderstorms erupt when the east coast breeze eventually collides with thunderstorm outflows from the west, but unexpected convection takes place prior to their merger along a well-defined confluence zone associated with a persistent quasi-stationary roll vortex signature. In this study, complementary interactions between roll vortex convergence lines and the sea-breeze front are not sufficient to trigger deep convection. However, organized convergence along the eastward-spreading thunderstorm outflows did interact periodically with roll vortex convergence maxima to initiate a new series of new storms. Results from two-dimensional numerical model simulations replicate many of the observed boundary layer features. Surface heating produces circulations similar to sea-breeze frontal zones that appear near the coastlines and progress steadily toward each other as the interior boundary layer deepens. Vertical velocity maxima develop over the associated convergence zones, but weaker periodic maxima also occur within the interior air mass at intervals similar to the spacing of observed horizontal roll vortices.

  5. Dynamin at actin tails.

    PubMed

    Lee, Eunkyung; De Camilli, Pietro

    2002-01-01

    Dynamin, the product of the shibire gene of Drosophila, is a GTPase critically required for endocytosis. Some studies have suggested a functional link between dynamin and the actin cytoskeleton. This link is of special interest, because there is evidence implicating actin dynamics in endocytosis. Here we show that endogenous dynamin 2, as well as green fluorescence protein fusion proteins of both dynamin 1 and 2, is present in actin comets generated by Listeria or by type I PIP kinase (PIPK) overexpression. In PIPK-induced tails, dynamin is further enriched at the interface between the tails and the moving organelles. Dynamin mutants harboring mutations in the GTPase domain inhibited nucleation of actin tails induced by PIPK and moderately reduced their speed. Although dynamin localization to the tails required its proline-rich domain, expression of a dynamin mutant lacking this domain also diminished tail formation. In addition, this mutant disrupted a membrane-associated actin scaffold (podosome rosette) previously shown to include dynamin. These findings suggest that dynamin is part of a protein network that controls nucleation of actin from membranes. At endocytic sites, dynamin may couple the fission reaction to the polymerization of an actin pool that functions in the separation of the endocytic vesicles from the plasma membrane. PMID:11782545

  6. Force generation by endocytic actin patches in budding yeast.

    PubMed

    Carlsson, Anders E; Bayly, Philip V

    2014-04-15

    Membrane deformation during endocytosis in yeast is driven by local, templated assembly of a sequence of proteins including polymerized actin and curvature-generating coat proteins such as clathrin. Actin polymerization is required for successful endocytosis, but it is not known by what mechanisms actin polymerization generates the required pulling forces. To address this issue, we develop a simulation method in which the actin network at the protein patch is modeled as an active gel. The deformation of the gel is treated using a finite-element approach. We explore the effects and interplay of three different types of force driving invagination: 1), forces perpendicular to the membrane, generated by differences between actin polymerization rates at the edge of the patch and those at the center; 2), the inherent curvature of the coat-protein layer; and 3), forces parallel to the membrane that buckle the coat protein layer, generated by an actomyosin contractile ring. We find that with optimistic estimates for the stall stress of actin gel growth and the shear modulus of the actin gel, actin polymerization can generate almost enough force to overcome the turgor pressure. In combination with the other mechanisms, actin polymerization can the force over the critical value.

  7. Force Generation by Endocytic Actin Patches in Budding Yeast

    PubMed Central

    Carlsson, Anders E.; Bayly, Philip V.

    2014-01-01

    Membrane deformation during endocytosis in yeast is driven by local, templated assembly of a sequence of proteins including polymerized actin and curvature-generating coat proteins such as clathrin. Actin polymerization is required for successful endocytosis, but it is not known by what mechanisms actin polymerization generates the required pulling forces. To address this issue, we develop a simulation method in which the actin network at the protein patch is modeled as an active gel. The deformation of the gel is treated using a finite-element approach. We explore the effects and interplay of three different types of force driving invagination: 1), forces perpendicular to the membrane, generated by differences between actin polymerization rates at the edge of the patch and those at the center; 2), the inherent curvature of the coat-protein layer; and 3), forces parallel to the membrane that buckle the coat protein layer, generated by an actomyosin contractile ring. We find that with optimistic estimates for the stall stress of actin gel growth and the shear modulus of the actin gel, actin polymerization can generate almost enough force to overcome the turgor pressure. In combination with the other mechanisms, actin polymerization can the force over the critical value. PMID:24739159

  8. Actin Mechanics and Fragmentation*

    PubMed Central

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

    2015-01-01

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

  9. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  10. Effect of single or combined climatic and hygienic stress in four layer lines: 1. Performance.

    PubMed

    Star, L; Kemp, B; van den Anker, I; Parmentier, H K

    2008-06-01

    Effects of long-term climatic stress (heat exposure), short-term hygienic stress [lipopolysaccharide (LPS)], or a combination of both challenges on performance of 4 layer lines were investigated. The lines were earlier characterized by natural humoral immune competence and survival rate. At 22 wk of age, 80 hens per line were randomly divided over 2 identical climate chambers and exposed to a constant high temperature (32 degrees C) or a control temperature (21 degrees C) for 23 d. Half of the hens housed in each chamber were i.v. injected with LPS at d 1 after the start of the heat stress period. The effect of heat, LPS, or a combined challenge on feed intake, BW, hen-day egg production, egg weight, and egg shell thickness were investigated. Feed intake, BW, hen-day egg production, egg weight, and egg shell thickness were significantly reduced by heat stress. Administration of LPS significantly reduced feed intake, BW (LPS x time interaction), hen-day egg production, and egg weight (LPS x time interaction). Hens were able to recover from LPS administration but did not completely adapt to heat stress. Hens still lost weight, had a lower feed intake and hen-day egg production after 23 d of continuous exposure to heat stress. These data suggest a different nature of short-term LPS exposure versus long-term heat exposure affecting performance parameters of laying hens, and different adaptation mechanisms of hens toward these stressors. Neither natural humoral immune competence nor survival rate, for which the lines had been earlier characterized, were indicative of the response to different stressors. However, significant line x heat interactions were found for feed intake and hen-day egg production, and a line x heat x time interaction for BW, whereas a line x LPS interaction was found for hen-day egg production and a line x LPS x time interaction for BW. The lines had similar response patterns, but differed in response levels, suggesting that some lines were better

  11. A leaky-wave antenna using double-layered metamaterial transmission line

    NASA Astrophysics Data System (ADS)

    Xu, He-Xiu; Wang, Guang-Ming; Qi, Mei-Qing

    2013-05-01

    A novel leaky-wave antenna (LWA) is proposed using a double-layered resonant-type metamaterial (MTM) transmission line (TL). The MTM TL is composed of periodically arranged complementary split ring resonators (CSRRs), capacitive gaps, and metal caps. By introducing the extra metal cap in additional layer of the basic artificial MTM TL element, an increased left handed capacitor by 36 % with respect to that using none cap is engineered, which is necessary to implement a balanced condition, and thus a continuous beam steering property of the resultant LWA in terms of providing phase constants from negative to positive values. For verification, a 20-cells LWA sample is fabricated and measured. Consistent numerical and experimental results have both validated the continuous frequency-scanning capabilities of the antenna from backward -29° to forward 72° (including the broadside). The proposed prescription opens a way toward new types of MTM LWAs with easily engineered broadside radiation.

  12. Sound transmission through double cylindrical shells lined with porous material under turbulent boundary layer excitation

    NASA Astrophysics Data System (ADS)

    Zhou, Jie; Bhaskar, Atul; Zhang, Xin

    2015-11-01

    This paper investigates sound transmission through double-walled cylindrical shell lined with poroelastic material in the core, excited by pressure fluctuations due to the exterior turbulent boundary layer (TBL). Biot's model is used to describe the sound wave propagating in the porous material. Three types of constructions, bonded-bonded, bonded-unbonded and unbonded-unbonded, are considered in this study. The power spectral density (PSD) of the inner shell kinetic energy is predicted for two turbulent boundary layer models, different air gap depths and three types of polyimide foams, respectively. The peaks of the inner shell kinetic energy due to shell resonance, hydrodynamic coincidence and acoustic coincidence are discussed. The results show that if the frequency band over the ring frequency is of interest, an air gap, even if very thin, should exist between the two elastic shells for better sound insulation. And if small density foam has a high flow resistance, a superior sound insulation can still be maintained.

  13. Actin Automata with Memory

    NASA Astrophysics Data System (ADS)

    Alonso-Sanz, Ramón; Adamatzky, Andy

    Actin is a globular protein which forms long polar filaments in eukaryotic. The actin filaments play the roles of cytoskeleton, motility units, information processing and learning. We model actin filament as a double chain of finite state machines, nodes, which take states “0” and “1”. The states are abstractions of absence and presence of a subthreshold charge on actin units corresponding to the nodes. All nodes update their state in parallel to discrete time. A node updates its current state depending on states of two closest neighbors in the node chain and two closest neighbors in the complementary chain. Previous models of actin automata consider momentary state transitions of nodes. We enrich the actin automata model by assuming that states of nodes depend not only on the current states of neighboring node but also on their past states. Thus, we assess the effect of memory of past states on the dynamics of acting automata. We demonstrate in computational experiments that memory slows down propagation of perturbations, decrease entropy of space-time patterns generated, transforms traveling localizations to stationary oscillators, and stationary oscillations to still patterns.

  14. Hypersonic stagnation line merged layer flow on blunt axisymmetric bodies of arbitrary shape

    NASA Technical Reports Server (NTRS)

    Jain, Amolak S.

    1993-01-01

    The problem of hypersonic stagnation line merged-layer flow of variously shaped blunt asisymmetric bodies is here formulated in such a way as to allow analytical calculations for bodies generated by a conic section. The governing equations encompass, apart from the usual parameters, the eccentricity of the conic section that generates the body-of-revolution for the effect of body shape on the solution obtained. The stagnation-point surface pressure increases as the favorable pressure gradient decreases, in the course of a change of body shape from spherical to hyperboloid.

  15. The Molecular Evolution of Actin

    PubMed Central

    Hightower, Robin C.; Meagher, Richard B.

    1986-01-01

    We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3–7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated <3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11–15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8–10%, whereas these members within the soybean actin gene family ranged from 6–9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence

  16. Direct Simulation of Evolution and Control of Nonlinear Instabilities in Attachment-Line Boundary Layers

    NASA Technical Reports Server (NTRS)

    Joslin, Ronald D.

    2004-01-01

    The unsteady, incompressible Navier-Stokes equations are used for the direct numerical simulation (DNS) of spatially evolving disturbances in a three-dimensional (3-D) attachment-line boundary layer. Two-dimensional (2-D) disturbances are introduced either by forcing at the in ow or by harmonic-source generators at the wall; 3-D disturbances are introduced by harmonic-source generators at the wall. The DNS results are in good agreement with both 2-D non-parallel theory (for small-amplitude disturbances) and weakly nonlinear theory (for finite-amplitude disturbances), which validates the two theories. The 2-D DNS results indicate that nonlinear disturbance growth occurs near branch II of the neutral stability curve; however, steady suction can be used to stabilize this disturbance growth. For 3-D instabilities that are generated o the attachment line, spreading both toward and away from the attachment line causes energy transfer to the attachment-line and downstream instabilities; suction stabilizes these instabilities. Furthermore, 3-D instabilities are more stable than 2-D or quasi-2-D instabilities.

  17. Flow in a wavy-walled channel lined with a poroelastic layer

    NASA Astrophysics Data System (ADS)

    Wei, H. H.; Waters, S. L.; Liu, S. Q.; Grotberg, J. B.

    2003-10-01

    Motivated by physiological flows in capillaries, venules and the pleural space, the pressure-driven flow of a Newtonian fluid in a two-dimensional wavy-walled channel is investigated theoretically. The sinusoidal wavy shape is due to the configuration of underlying cells, their nuclei and intercellular junctions or clefts. The walls are lined with a thin poroelastic layer that models the glycocalyx coating of the cell surface. The upper and lower wavy walls are offset axially by the phase angle Phi , where Phi { =} 0 (upi) yields an antisymmetric (symmetric) channel. Biphasic theory is employed for the poroelastic layer and the flow is solved by a lubrication approximation using a small parameter, delta {≪} 1, where delta is the channel width/wavelength ratio. The velocity fields in the core and layer are determined as perturbation expansions in delta(2) and finite-Reynolds-number effects occur at O(delta(2)) assuming delta(2) Re {≪} 1. When the hydraulic resistivity, alpha, the ratio of the channel width to the Darcy permeability, is sufficiently large and Phi is near enough to upi, the flow develops a trapped recirculation eddy within the glycocalyx layer near the widest part of the channel. This can be of significance to transport through the cellular boundary, since that location corresponds to intercellular clefts through which important fluid and solute exchange occurs. Increasing |Phi {-} upi | diminishes the recirculation region. Increasing the Reynolds number moves the recirculation slightly upstream. Both layer velocity and wall shear stresses decrease as alpha increases and support the appearance of flow recirculation. Further, the wavy geometry allows a portion of the flow to enter and exit the layer, which provides a mechanism for convective transport between these two regions that otherwise have only diffusive interactions. The relevant Péclet number is Pe {=} V(*_nb/D) where D is molecular diffusivity and V(*_n) is the normal velocity to the

  18. The split-cross-bridge resistor for measuring the sheet resistance, linewidth, and line spacing of conducting layers

    NASA Technical Reports Server (NTRS)

    Buehler, M. G.; Hershey, C. W.

    1986-01-01

    A new test structure was developed for evaluating the line spacing between conductors on the same layer using an electrical measurement technique. This compact structure can also be used to measure the sheet resistance, linewidth, and line pitch of the conducting layer. Using an integrated-circuit fabrication process, this structure was fabricated in diffused polycrystalline silicon and metal layers and measured optically and electrically. For the techniques used, the optical measurements were typically one-quarter micron greater than the electrical measurements. Most electrically measured line pitch values were within 2 percent of the designed value. A small difference between the measured and designed line pitch is used to validate sheet resistance, linewidth, and line spacing values.

  19. Spatially Developing Secondary Instabilities and Attachment Line Instability in Supersonic Boundary Layers

    NASA Technical Reports Server (NTRS)

    Li, Fei; Choudhari, Meelan M.

    2008-01-01

    This paper reports on progress towards developing a spatial stability code for compressible shear flows with two inhomogeneous directions, such as crossflow dominated swept-wing boundary layers and attachment line flows. Certain unique aspects of formulating a spatial, two-dimensional eigenvalue problem for the secondary instability of finite amplitude crossflow vortices are discussed. A primary test case used for parameter study corresponds to the low-speed, NLF-0415(b) airfoil configuration as tested in the ASU Unsteady Wind Tunnel, wherein a spanwise periodic array of roughness elements was placed near the leading edge in order to excite stationary crossflow modes with a specified fundamental wavelength. The two classes of flow conditions selected for this analysis include those for which the roughness array spacing corresponds to either the naturally dominant crossflow wavelength, or a subcritical wavelength that serves to reduce the growth of the naturally excited dominant crossflow modes. Numerical predictions are compared with the measured database, both as indirect validation for the spatial instability analysis and to provide a basis for comparison with a higher Reynolds number, supersonic swept-wing configuration. Application of the eigenvalue analysis to the supersonic configuration reveals that a broad spectrum of stationary crossflow modes can sustain sufficiently strong secondary instabilities as to potentially cause transition over this configuration. Implications of this finding for transition control in swept wing boundary layers are examined. Finally, extension of the spatial stability analysis to supersonic attachment line flows is also considered.

  20. Simulation of Nonlinear Instabilities in an Attachment-Line Boundary Layer

    NASA Technical Reports Server (NTRS)

    Joslin, Ronald D.

    1996-01-01

    The linear and the nonlinear stability of disturbances that propagate along the attachment line of a three-dimensional boundary layer is considered. The spatially evolving disturbances in the boundary layer are computed by direct numerical simulation (DNS) of the unsteady, incompressible Navier-Stokes equations. Disturbances are introduced either by forcing at the in ow or by applying suction and blowing at the wall. Quasi-parallel linear stability theory and a nonparallel theory yield notably different stability characteristics for disturbances near the critical Reynolds number; the DNS results con rm the latter theory. Previously, a weakly nonlinear theory and computations revealed a high wave-number region of subcritical disturbance growth. More recent computations have failed to achieve this subcritical growth. The present computational results indicate the presence of subcritically growing disturbances; the results support the weakly nonlinear theory. Furthermore, an explanation is provided for the previous theoretical and computational discrepancy. In addition, the present results demonstrate that steady suction can be used to stabilize disturbances that otherwise grow subcritically along the attachment line.

  1. Vortex dynamics of in-line twin synthetic jets in a laminar boundary layer

    NASA Astrophysics Data System (ADS)

    Wen, Xin; Tang, Hui; Duan, Fei

    2015-08-01

    An experimental investigation is conducted on the vortices induced by twin synthetic jets (SJs) in line with a laminar boundary layer flow over a flat plate. The twin SJs operating at four different phase differences, i.e., Δϕ = 0°, 90°, 180°, and 270°, are visualized using a stereoscopic color dye visualization system and measured using a two-dimensional particle image velocimetry (PIV) system. It is found that depending on the phase difference of twin SJs, three types of vortex structures are produced. At Δϕ = 90°, the two hairpin vortices interact in a very constructive way in terms of the vortex size, strength, and celerity, forming one combined vortex. At Δϕ = 270°, the two individual hairpin vortices do not have much interaction, forming two completely separated hairpin vortices that behave like doubling the frequency of the single SJ case. At Δϕ = 0° and 180°, the two hairpin vortices produced by the twin SJ actuators are close enough, with the head of one hairpin vortex coupled with the legs of the other, forming partially interacting vortex structures. Quantitative analysis of the twin SJs is conducted, including the time histories of vortex circulation in the mid-span plane as well as a selected spanwise-wall-normal plane, and the influence of the twin SJs on the boundary layer flow filed. In addition, dynamic mode decomposition analysis of the PIV data is conducted to extract representative coherent structures. Through this study, a better understanding in the vortex dynamics associated with the interaction of in-line twin SJs in laminar boundary layers is achieved, which provides useful information for future SJ-array applications.

  2. Single-layer and bilayer graphene superlattices: collimation, additional Dirac points and Dirac lines.

    PubMed

    Barbier, Michaël; Vasilopoulos, Panagiotis; Peeters, François M

    2010-12-13

    We review the energy spectrum and transport properties of several types of one-dimensional superlattices (SLs) on single-layer and bilayer graphene. In single-layer graphene, for certain SL parameters an electron beam incident on an SL is highly collimated. On the other hand, there are extra Dirac points generated for other SL parameters. Using rectangular barriers allows us to find analytical expressions for the location of new Dirac points in the spectrum and for the renormalization of the electron velocities. The influence of these extra Dirac points on the conductivity is investigated. In the limit of δ-function barriers, the transmission T through and conductance G of a finite number of barriers as well as the energy spectra of SLs are periodic functions of the dimensionless strength P of the barriers, Pδ(x) = V(x)/ħv(F), with v(F) the Fermi velocity. For a Kronig-Penney SL with alternating sign of the height of the barriers, the Dirac point becomes a Dirac line for P = π/2+nπ with n an integer. In bilayer graphene, with an appropriate bias applied to the barriers and wells, we show that several new types of SLs are produced and two of them are similar to type I and type II semiconductor SLs. Similar to single-layer graphene SLs, extra 'Dirac' points are found in bilayer graphene SLs. Non-ballistic transport is also considered. PMID:21041227

  3. Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration

    PubMed Central

    Lu, Rong; Bian, Fang; Lin, Jing; Su, Zhitao; Qu, Yangluowa; Pflugfelder, Stephen C.; Li, De-Quan

    2012-01-01

    There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5–14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1×104 in a 35-mm dish (9.6 cm2) grew to confluence (about 1.87–2.41×106 cells) in 12–14 days, representing 187–241 fold expansion with over 7–8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction. PMID:22723892

  4. Intranuclear Actin Regulates Osteogenesis

    PubMed Central

    Sen, Buer; Xie, Zhihui; Uzer, Gunes; Thompson, William R.; Styner, Maya; Wu, Xin; Rubin, Janet

    2016-01-01

    Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone. PMID:26140478

  5. The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages.

    PubMed

    Hartwig, J H; Shevlin, P

    1986-09-01

    A highly branched filament network is the principal structure in the periphery of detergent-extracted cytoskeletons of macrophages that have been spread on a surface and either freeze or critical point dried, and then rotary shadowed with platinum-carbon. This array of filaments completely fills lamellae extended from the cell and bifurcates to form 0.2-0.5 micron thick layers on the top and bottom of the cell body. Reaction of the macrophage cytoskeletons with anti-actin IgG and with anti-IgG bound to colloidal gold produces dense staining of these filaments, and incubation with myosin subfragment 1 uniformly decorates these filaments, identifying them as actin. 45% of the total cellular actin and approximately 70% of actin-binding protein remains in the detergent-insoluble cell residue. The soluble actin is not filamentous as determined by sedimentation analysis, the DNAase I inhibition assay, and electron microscopy, indicating that the cytoskeleton is not fragmented by detergent extraction. The spacing between the ramifications of the actin network is 94 +/- 47 nm and 118 +/- 72 nm in cytoskeletons prepared for electron microscopy by freeze drying and critical point drying, respectively. Free filament ends are rare, except for a few which project upward from the body of the network or which extend down to the substrate. Filaments of the network intersect predominantly at right angles to form either T-shaped and X-shaped overlaps having striking perpendicularity or else Y-shaped intersections composed of filaments intersecting at 120-130 degrees angles. The actin filament concentration in the lamellae is high, with an average value of 12.5 mg/ml. The concentration was much more uniform in freeze-dried preparations than in critical point-dried specimens, indicating that there is less collapse associated with the freezing technique. The orthogonal actin network of the macrophage cortical cytoplasm resembles actin gels made with actin-binding protein. Reaction of

  6. Simulation of Three-Dimensional Symmetric and Asymmetric Instabilities in Attachment-Line Boundary Layers

    NASA Technical Reports Server (NTRS)

    Joslin, Ronald D.

    1996-01-01

    On a swept wing, contamination along the leading edge, Tollmien-Schlichting waves, stationary or traveling crossflow vortices, and/or Taylor-Gortler vortices can cause the catastrophic breakdown of laminar to turbulent flow, which leads to increased skin-friction drag for the aircraft. The discussion in this Note will be limited to disturbances which evolve along the attachment line (leading edge of swept wing). If the Reynolds number of the attachment-line boundary layer is greater than some critical value, then the complete wing is inevitably engulfed in turbulent flow. Essentially, there are two critical Reynolds number points that must be considered. The first is for small-amplitude disturbances, and the second is for bypass transition. The present study will use direct numerical simulations to validate a linear 2D-eigenvalue prediction method based on parabolized stability equations by Lin and Malik. This method is considered because it suggests that a number of symmetric and asymmetric modes exist and are stable or unstable on the attachment line depending on the Reynolds number. If validated, the approach would predict a number of modes which are linearly damped in the Reynolds number regime 100 to 245; however, these modes may grow nonlinearly and provide an explanation to this region.

  7. Measuring F-actin properties in dendritic spines

    PubMed Central

    Koskinen, Mikko; Hotulainen, Pirta

    2014-01-01

    During the last decade, numerous studies have demonstrated that the actin cytoskeleton plays a pivotal role in the control of dendritic spine shape. Synaptic stimulation rapidly changes the actin dynamics and many actin regulators have been shown to play roles in neuron functionality. Accordingly, defects in the regulation of the actin cytoskeleton in neurons have been implicated in memory disorders. Due to the small size of spines, it is difficult to detect changes in the actin structures in dendritic spines by conventional light microscopy imaging. Instead, to know how tightly actin filaments are bundled together, and how fast the filaments turnover, we need to use advanced microscopy techniques, such as fluorescence recovery after photobleaching (FRAP), photoactivatable green fluorescent protein (PAGFP) fluorescence decay and fluorescence anisotropy. Fluorescence anisotropy, which measures the Förster resonance energy transfer (FRET) between two GFP fluorophores, has been proposed as a method to measure the level of actin polymerization. Here, we propose a novel idea that fluorescence anisotropy could be more suitable to study the level of actin filament bundling instead of actin polymerization. We validate the method in U2OS cell line where the actin structures can be clearly distinguished and apply to analyze how actin filament organization in dendritic spines changes during neuronal maturation. In addition to fluorescence anisotropy validation, we take a critical look at the properties and limitations of FRAP and PAGFP fluorescence decay methods and offer our proposals for the analysis methods for these approaches. These three methods complement each other, each providing additional information about actin dynamics and organization in dendritic spines. PMID:25140131

  8. Determination of nitrogen to carbon abundance ratios from transition layer emission lines

    NASA Technical Reports Server (NTRS)

    Boehm-Vitense, Erika

    1992-01-01

    We have finished studying the nitrogen to carbon abundance ratios for stars with different effective temperatures T(sub eff) and luminosities using transition layer emission lines and using spectra available in the IUE archives. The N/C abundance ratio determinations using transition layer emission lines are as accurate as the photospheric abundance determinations as found by comparison of results obtained by both methods for the same stars. Our measurements confirm photospheric abundance determinations in regions of the HR diagram where they can be obtained. Our studies have extended the temperature range to higher temperatures. They have shown the exact positions in the HR diagram where the mixing due to the outer convection zones reaches deep enough to bring nuclear processed material to the surface. This occurs at effective temperatures which are higher by delta log T(sub eff) approximately 0.04 or roughly 400 K than expected theoretically. Since the depth of the convection zone increases rapidly with decreasing T(sub eff) this may indicate considerable overshoot beyond the lower boundary of the convection zone. Our N/C abundance ratio determinations from transition layer emission lines have confirmed that the actual enrichment observed for some cool giants is larger than expected theoretically, again indicating a larger degree of mixing in several stars either from below or from above. For the supergiants it probably indicates overshoot above the convective core in the progenitor main sequence stars. For the more massive giants this may also be the case, though we did not find a correlation between delta log N/C and the absolute magnitudes, but these are rather uncertain. As byproducts of these studies we also found anomalies in Si/C and N/C abundance ratios for F giants which can be understood as the relict of surface abundance changes for their main sequence progenitors due to diffusion. This anomaly disappears for G giants, for which the depths of the

  9. Magnetic field line reconnection experiments. V - Current disruptions and double layers

    NASA Technical Reports Server (NTRS)

    Stenzel, R. L.; Gekelman, W.; Wild, N.

    1983-01-01

    An investigation is conducted of the stability of a large laboratory plasma current sheet, which has been generated in the process of magnetic field line reconnection, with respect to local current increases. Magnetic flux variations in regions remote from the current sheet generate an inductive voltage in the current loop that drops off inside the plasma in the form of a potential double layer, leading to particle acceleration with velocities much larger than those expected from the steady state electric fields in the plasma. A model for the mechanism of the current disruptions is formulated in which the potential structure leads to ion expulsion, creating a localized density drop. The associated current drop in an inductive circuit drives the potential structure, providing feedback for the disruptive instability. Similarities to, and differences from, magnetospheric substorm phenomena are noted.

  10. Excitation of Love waves in a thin film layer by a line source.

    NASA Technical Reports Server (NTRS)

    Tuan, H.-S.; Ponamgi, S. R.

    1972-01-01

    The excitation of a Love surface wave guided by a thin film layer deposited on a semiinfinite substrate is studied in this paper. Both the thin film and the substrate are considered to be elastically isotropic. Amplitudes of the surface wave in the thin film region and the substrate are found in terms of the strength of a line source vibrating in a direction transverse to the propagating wave. In addition to the surface wave, the bulk shear wave excited by the source is also studied. Analytical expressions for the bulk wave amplitude as a function of the direction of propagation, the acoustic powers transported by the surface and bulk waves, and the efficiency of surface wave excitation are obtained. A numerical example is given to show how the bulk wave radiation pattern depends upon the source frequency, the film thickness and other important parameters of the problem. The efficiency of surface wave excitation is also calculated for various parameter values.

  11. Direct Simulation of Evolution and Control of Three-Dimensional Instabilities in Attachment-Line Boundary Layers

    NASA Technical Reports Server (NTRS)

    Joslin, Ronald D.

    1995-01-01

    The spatial evolution of three-dimensional disturbances in an attachment-line boundary layer is computed by direct numerical simulation of the unsteady, incompressible Navier-Stokes equations. Disturbances are introduced into the boundary layer by harmonic sources that involve unsteady suction and blowing through the wall. Various harmonic- source generators are implemented on or near the attachment line, and the disturbance evolutions are compared. Previous two-dimensional simulation results and nonparallel theory are compared with the present results. The three-dimensional simulation results for disturbances with quasi-two-dimensional features indicate growth rates of only a few percent larger than pure two-dimensional results; however, the results are close enough to enable the use of the more computationally efficient, two-dimensional approach. However, true three-dimensional disturbances are more likely in practice and are more stable than two-dimensional disturbances. Disturbances generated off (but near) the attachment line spread both away from and toward the attachment line as they evolve. The evolution pattern is comparable to wave packets in at-plate boundary-layer flows. Suction stabilizes the quasi-two-dimensional attachment-line instabilities, and blowing destabilizes these instabilities; these results qualitatively agree with the theory. Furthermore, suction stabilizes the disturbances that develop off the attachment line. Clearly, disturbances that are generated near the attachment line can supply energy to attachment-line instabilities, but suction can be used to stabilize these instabilities.

  12. Amplification of actin polymerization forces

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  14. Coupling of the hydration water dynamics and the internal dynamics of actin detected by quasielastic neutron scattering

    SciTech Connect

    Fujiwara, Satoru; Plazanet, Marie; Oda, Toshiro

    2013-02-15

    Highlights: ► Quasielastic neutron scattering spectra of F-actin and G-actin were measured. ► Analysis of the samples in D{sub 2}O and H{sub 2}O provided the spectra of hydration water. ► The first layer hydration water around F-actin is less mobile than around G-actin. ► This difference in hydration water is in concert with the internal dynamics of actin. ► Water outside the first layer behaves bulk-like but influenced by the first layer. -- Abstract: In order to characterize dynamics of water molecules around F-actin and G-actin, quasielastic neutron scattering experiments were performed on powder samples of F-actin and G-actin, hydrated either with D{sub 2}O or H{sub 2}O, at hydration ratios of 0.4 and 1.0. By combined analysis of the quasielastic neutron scattering spectra, the parameter values characterizing the dynamics of the water molecules in the first hydration layer and those of the water molecules outside of the first layer were obtained. The translational diffusion coefficients (D{sub T}) of the hydration water in the first layer were found to be 1.2 × 10{sup −5} cm{sup 2}/s and 1.7 × 10{sup −5} cm{sup 2}/s for F-actin and G-actin, respectively, while that for bulk water was 2.8 × 10{sup −5} cm{sup 2}/s. The residence times were 6.6 ps and 5.0 ps for F-actin and G-actin, respectively, while that for bulk water was 0.62 ps. These differences between F-actin and G-actin, indicating that the hydration water around G-actin is more mobile than that around F-actin, are in concert with the results of the internal dynamics of F-actin and G-actin, showing that G-actin fluctuates more rapidly than F-actin. This implies that the dynamics of the hydration water is coupled to the internal dynamics of the actin molecules. The D{sub T} values of the water molecules outside of the first hydration layer were found to be similar to that of bulk water though the residence times are strongly affected by the first hydration layer. This supports the

  15. IFT88 influences chondrocyte actin organization and biomechanics

    PubMed Central

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

    2016-01-01

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

  16. Control of actin-based motility through localized actin binding.

    PubMed

    Banigan, Edward J; Lee, Kun-Chun; Liu, Andrea J

    2013-12-01

    A wide variety of cell biological and biomimetic systems use actin polymerization to drive motility. It has been suggested that an object such as a bacterium can propel itself by self-assembling a high concentration of actin behind it, if it is repelled by actin. However, it is also known that it is essential for the moving object to bind actin. Therefore, a key question is how the actin tail can propel an object when it both binds and repels the object. We present a physically consistent Brownian dynamics model for actin-based motility that includes the minimal components of the dendritic nucleation model and allows for both attractive and repulsive interactions between actin and a moveable disc. We find that the concentration gradient of filamentous actin generated by polymerization is sufficient to propel the object, even with moderately strong binding interactions. Additionally, actin binding can act as a biophysical cap, and may directly control motility through modulation of network growth. Overall, this mechanism is robust in that it can drive motility against a load up to a stall pressure that depends on the Young's modulus of the actin network and can explain several aspects of actin-based motility.

  17. Auxin Stimulates Its Own Transport by Shaping Actin Filaments1

    PubMed Central

    Nick, Peter; Han, Min-Jung; An, Gyeunhung

    2009-01-01

    The directional transport of the plant hormone auxin has been identified as central element of axis formation and patterning in plants. This directionality of transport depends on gradients, across the cell, of auxin-efflux carriers that continuously cycle between plasma membrane and intracellular compartments. This cycling has been proposed to depend on actin filaments. However, the role of actin for the polarity of auxin transport has been disputed. The organization of actin, in turn, has been shown to be under control of auxin. By overexpression of the actin-binding protein talin, we have generated transgenic rice (Oryza sativa) lines, where actin filaments are bundled to variable extent and, in consequence, display a reduced dynamics. We show that this bundling of actin filaments correlates with impaired gravitropism and reduced longitudinal transport of auxin. We can restore a normal actin configuration by addition of exogenous auxins and restore gravitropism as well as polar auxin transport. This rescue is mediated by indole-3-acetic acid and 1-naphthyl acetic acid but not by 2,4-dichlorophenoxyacetic acid. We interpret these findings in the context of a self-referring regulatory circuit between polar auxin transport and actin organization. This circuit might contribute to the self-amplification of auxin transport that is a central element in current models of auxin-dependent patterning. PMID:19633235

  18. Guardians of the actin monomer.

    PubMed

    Xue, Bo; Robinson, Robert C

    2013-01-01

    Actin is a universal force provider in eukaryotic cells. Biological processes harness the pressure generated from actin polymerization through dictating the time, place and direction of filament growth. As such, polymerization is initiated and maintained via tightly controlled filament nucleation and elongation machineries. Biological systems integrate force into their activities through recruiting and activating these machineries. In order that actin function as a common force generating polymerization motor, cells must maintain a pool of active, polymerization-ready monomeric actin, and minimize extemporaneous polymerization. Maintenance of the active monomeric actin pool requires the recycling of actin filaments, through depolymerization, nucleotide exchange and reloading of the polymerization machineries, while the levels of monomers are constantly monitored and supplemented, when needed, via the access of a reserve pool of monomers and through gene expression. Throughout its monomeric life, actin needs to be protected against gratuitous nucleation events. Here, we review the proteins that act as custodians of monomeric actin. We estimate their levels on a tissue scale, and calculate the implied concentrations of each actin complex based on reported binding affinities. These estimations predict that monomeric actin is rarely, if ever, alone. Thus, the guardians keep the volatility of actin in check, so that its explosive power is only released in the controlled environments of the nucleation and polymerization machineries. PMID:24268205

  19. Modeling of Ice Flow and Internal Layers Along a Flow Line Through Swiss Camp in West Greenland

    NASA Technical Reports Server (NTRS)

    Wang, W. L.; Zwally, H. Jay; Abdalati, W.; Luo, S.; Koblinsky, Chester J. (Technical Monitor)

    2001-01-01

    An anisotropic ice flow line model is applied to a flow line through Swiss Camp (69.57 N, 49.28 W) in West Greenland to estimate the dates of internal layers detected by Radio-Echo Sounding measurements. The effect of an anisotropic ice fabric on ice flow is incorporated into the steady state flow line model. The stress-strain rate relationship for anisotropic ice is characterized by an enhancement factor based on the laboratory observations of ice deformation under combined compression and shear stresses. By using present-day data of accumulation rate, surface temperature, surface elevation and ice thickness along the flow line as model inputs, a very close agreement is found between the isochrones generated from the model and the observed internal layers with confirmed dates. The results indicate that this part of Greenland ice sheet is primarily in steady state.

  20. Instability suppression in viscoelastic film flows down an inclined plane lined with a deformable solid layer.

    PubMed

    Jain, Aashish; Shankar, V

    2007-10-01

    The linear stability of viscoelastic (Oldroyd-B) film flow down an inclined plane lined with a deformable (neo-Hookean) solid layer is analyzed using low-wave-number asymptotic analysis and the Chebyshev-Tau spectral numerical method. The free surface of film flows of viscoelastic liquids, unlike that of their Newtonian counterparts, becomes unstable in flow down a rigid inclined surface even in the absence of fluid inertia, due to the elastic nature of the liquids. For film flow past a deformable solid, our low-wave-number asymptotic analysis reveals that the solid deformability has a stabilizing effect on the free-surface instability, and, remarkably, this prediction is insensitive to rheology of the liquid film, be it viscoelastic or Newtonian. Using the spectral numerical method, we demonstrate that the free-surface instability can be completely suppressed at all wave numbers when the solid becomes sufficiently deformable. For the case of pure polymeric liquids without any solvent, when the solid layer is made further deformable, both the free surface and the liquid-solid interface are destabilized at finite wave numbers. We also demonstrate a type of mode exchange phenomenon between the modes corresponding to the two interfaces. Importantly, our numerical results show that there is a sufficient range of shear modulus of the solid where both the modes are stable at all wave numbers. For polymer solutions described by the Oldroyd-B model, while the free-surface instability is suppressed by the deformable solid, a host of new unstable modes appear at finite Reynolds number and wave number because of the coupling between liquid flow and free shear waves in the solid. Our study thus demonstrates that the elastohydrodynamic coupling between liquid flow and solid deformation can be exploited either to suppress the free-surface instability (present otherwise in rigid inclines) in viscoelastic film flows, or to induce new instabilities that are absent in flow adjacent

  1. Nanosecond electric pulses trigger actin responses in plant cells

    SciTech Connect

    Berghoefer, Thomas; Eing, Christian; Flickinger, Bianca; Hohenberger, Petra; Wegner, Lars H.; Frey, Wolfgang; Nick, Peter

    2009-09-25

    We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.

  2. The Switch-associated Protein 70 (SWAP-70) Bundles Actin Filaments and Contributes to the Regulation of F-actin Dynamics*

    PubMed Central

    Chacón-Martínez, Carlos Andrés; Kiessling, Nadine; Winterhoff, Moritz; Faix, Jan; Müller-Reichert, Thomas; Jessberger, Rolf

    2013-01-01

    Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks. PMID:23921380

  3. Tungsten Contact and Line Resistance Reduction with Advanced Pulsed Nucleation Layer and Low Resistivity Tungsten Treatment

    NASA Astrophysics Data System (ADS)

    Chandrashekar, Anand; Chen, Feng; Lin, Jasmine; Humayun, Raashina; Wongsenakhum, Panya; Chang, Sean; Danek, Michal; Itou, Takamasa; Nakayama, Tomoo; Kariya, Atsushi; Kawaguchi, Masazumi; Hizume, Shunichi

    2010-09-01

    This paper describes electrical testing results of new tungsten chemical vapor deposition (CVD-W) process concepts that were developed to address the W contact and bitline scaling issues on 55 nm node devices. Contact resistance (Rc) measurements in complementary metal oxide semiconductor (CMOS) devices indicate that the new CVD-W process for sub-32 nm and beyond - consisting of an advanced pulsed nucleation layer (PNL) combined with low resistivity tungsten (LRW) initiation - produces a 20-30% drop in Rc for diffused NiSi contacts. From cross-sectional bright field and dark field transmission electron microscopy (TEM) analysis, such Rc improvement can be attributed to improved plugfill and larger in-feature W grain size with the advanced PNL+LRW process. More experiments that measured contact resistance for different feature sizes point to favorable Rc scaling with the advanced PNL+LRW process. Finally, 40% improvement in line resistance was observed with this process as tested on 55 nm embedded dynamic random access memory (DRAM) devices, confirming that the advanced PNL+LRW process can be an effective metallization solution for sub-32 nm devices.

  4. Contaminant Dispersion from a Line Source in a Turbulent Boundary Layer.

    NASA Astrophysics Data System (ADS)

    Chiang, K.-F.; Ong, L.; Wallace, J.; Bernard, P.

    1996-11-01

    Experiments of smoke dispersion from a line source in a turbulent boundary layer have been performed in a low-speed wind tunnel. To illuminate the smoke a laser light sheet, oriented in the x-y plane, was created by a copper-vapor plused laser with a variety of optics. The evolving smoke plume was photographed, synchronously with 4-sensor hot-wire probe data, on 16 mm film with a high speed movie camera. These digitized photographs constitute an ensemble of images with grey levels which were subsequently mapped into quantitative values of concentration by means of a calibration and transformation procedure. Combining the instantaneous concentration fluctuation and velocity fluctuation component values yielded the scalar fluxes \\overlineuc and \\overlinevc. Comparisons will be made between the experimental data and results from a channel flow DNS, as well as from model predictions. An image reconstruction of the 3 D plume structures from a set of y-z plane 2 D images, rapidly scanned in the streamwise x-direction, was carried out. Results from spatial and space-time correlations of the concentration fields, as well as conditional ensemble averages will be discussed.

  5. Dynacortin is a novel actin bundling protein that localizes to dynamic actin structures.

    PubMed

    Robinson, Douglas N; Ocon, Stephani S; Rock, Ronald S; Spudich, James A

    2002-03-15

    Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7-nm Stokes radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 microm K(d). Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 s. These results indicate that cells have evolved different actin-filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes.

  6. A Structural Basis for Regulation of Actin Polymerization by Pectenotoxins

    PubMed Central

    Allingham, John S.; Miles, Christopher O.; Rayment, Ivan

    2007-01-01

    Pectenotoxins (PTXs) are polyether macrolides found in certain dinoflagellates, sponges and shellfish, and have been associated with diarrhetic shellfish poisoning. In addition to their in vivo toxicity, some PTXs are potently cytotoxic in human cancer cell lines. Recent studies have demonstrated that disruption of the actin cytoskeleton may be a key function of these compounds, although no clarification their mechanism of action at a molecular level was available. We have obtained an X-ray crystal structure of PTX-2 bound to actin which, in combination with analyses of the effect of PTX-2 on purified actin filament dynamics, provides a molecular explanation for its effects on actin. PTX-2 formed a 1:1 complex with actin and engaged a novel site between subdomains 1 and 3. Based on models of the actin filament, PTX binding would disrupt key lateral contacts between the PTX-bound actin monomer and the lower lateral actin monomer within the filament, thereby capping the barbed-end. The location of this binding position within the interior of the filament indicates that it may not be accessible once polymerization has occurred, a hypothesis supported by our observation that PTX-2 caused filament capping without inducing filament severing. This mode of action is unique, as other actin filament destabilizing toxins appear to exclusively disrupt longitudinal monomer contacts allowing many of them to sever filaments in addition to capping them. Examination of the PTX-binding site on actin provides a rationalization for the structure–activity relationships observed in vivo and in vitro, and may provide a basis for predicting toxicity of PTX analogues. PMID:17599353

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

    PubMed

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

    2015-11-01

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

  8. Concentration profiles of actin-binding molecules in lamellipodia

    NASA Astrophysics Data System (ADS)

    Falcke, Martin

    2016-04-01

    Motile cells form lamellipodia in the direction of motion, which are flat membrane protrusions containing an actin filament network. The network flows rearward relative to the leading edge of the lamellipodium due to actin polymerization at the front. Thus, actin binding molecules are subject to transport towards the rear of the cell in the bound state and diffuse freely in the unbound state. We analyze this reaction-diffusion-advection process with respect to the concentration profiles of these species and provide an analytic approximation for them. Network flow may cause a depletion zone of actin binding molecules close to the leading edge. The existence of such zone depends on the free molecule concentration in the cell body, on the ratio of the diffusion length to the distance bound molecules travel rearward with the flow before dissociating, and the ratio of the diffusion length to the width of the region with network flow and actin binding. Our calculations suggest the existence of depletion zones for the F-actin cross-linkers filamin and α-actinin in fish keratocytes (and other cell types), which is in line with the small elastic moduli of the F-actin network close to the leading edge found in measurements of the force motile cells are able to exert.

  9. Dark-line atomic resonances in a submicron-thin Rb vapor layer

    NASA Astrophysics Data System (ADS)

    Sargsyan, A.; Sarkisyan, D.; Papoyan, A.

    2006-03-01

    We report an experimental investigation of the effect of electromagnetically induced transparency (EIT) using bichromatic laser radiation and an extremely thin cell filled with pure Rb with smoothly controllable thickness L of the atomic vapor layer in the range ˜780-1600nm , for which L is comparable to the laser wavelength λ resonant with the D2 line (780nm) . It is revealed that the transmission spectrum of the probe laser contains two sub-Doppler peaks that have different linewidths. The narrow peak corresponds to EIT, whereas the broader one results from velocity-selective optical pumping (VSOP) and repumping processes. It is demonstrated that in the case of nonzero detuning of the coupling laser, the EIT resonance and VSOP are shifted with respect to each other on the frequency scale, which makes it possible to observe a competition between the two effects (this is not possible to realize in an ordinary cell). Also, the Dicke-type coherent narrowing effect depending on the ratio L/λ influences the absorption spectrum of the probe laser. Formation of an EIT resonance is substantially favored for the atoms with slow normal velocity, caused by their longer interaction time with the bichromatic laser field. As a result of the predominant contribution of these atoms, the observed linewidth of the EIT resonance is only ˜9MHz , which is more than ten times narrower than the inverse window-to-window flight time. In an external magnetic field, three and five EIT resonances have been observed for the vapor thickness of L=2λ , on Rb87 and Rb85 , respectively.

  10. X-ray recordings reveal how a human disease-linked skeletal muscle α-actin mutation leads to contractile dysfunction.

    PubMed

    Ochala, Julien; Ravenscroft, Gianina; McNamara, Elyshia; Nowak, Kristen J; Iwamoto, Hiroyuki

    2015-12-01

    In humans, mutant skeletal muscle α-actin proteins are associated with contractile dysfunction, skeletal muscle weakness and a wide range of primarily skeletal muscle diseases. Despite this knowledge, the exact molecular mechanisms triggering the contractile dysfunction remain unknown. Here, we aimed to unravel these. Hence, we used a transgenic mouse model expressing a well-described D286G mutant skeletal muscle α-actin protein and recapitulating the human condition of contractile deregulation and severe skeletal muscle weakness. We then recorded and analyzed the small-angle X-ray diffraction patterns of isolated membrane-permeabilized myofibers. Results showed that upon addition of Ca(2+), the intensity changes of the second (1/19 nm(-1)) and sixth (1/5.9 nm(-1)) actin layer lines and of the first myosin meridional reflection (1/14.3 nm(-1)) were disrupted when the thin-thick filament overlap was optimal (sarcomere length of 2.5-2.6 μm). However these reflections were normal when the thin and thick filaments were not interacting (sarcomere length>3.6 μm). These findings demonstrate, for the first time, that the replacement of just one amino acid in the skeletal muscle α-actin protein partly prevents actin conformational changes during activation, disrupting the strong binding of myosin molecules. This leads to a limited myosin-related tropomyosin movement over the thin filaments, further affecting the amount of cross-bridges, explaining the contractile dysfunction. PMID:26407659

  11. Analysis of actinic flux profiles measured from an ozonesonde balloon

    NASA Astrophysics Data System (ADS)

    Wang, P.; Allaart, M.; Knap, W. H.; Stammes, P.

    2015-04-01

    A green light sensor has been developed at KNMI to measure actinic flux profiles using an ozonesonde balloon. In total, 63 launches with ascending and descending profiles were performed between 2006 and 2010. The measured uncalibrated actinic flux profiles are analysed using the Doubling-Adding KNMI (DAK) radiative transfer model. Values of the cloud optical thickness (COT) along the flight track were taken from the Spinning Enhanced Visible and Infrared Imager (SEVIRI) Cloud Physical Properties (CPP) product. The impact of clouds on the actinic flux profile is evaluated on the basis of the cloud modification factor (CMF) at the cloud top and cloud base, which is the ratio between the actinic fluxes for cloudy and clear-sky scenes. The impact of clouds on the actinic flux is clearly detected: the largest enhancement occurs at the cloud top due to multiple scattering. The actinic flux decreases almost linearly from cloud top to cloud base. Above the cloud top the actinic flux also increases compared to clear-sky scenes. We find that clouds can increase the actinic flux to 2.3 times the clear-sky value at cloud top and decrease it to about 0.05 at cloud base. The relationship between CMF and COT agrees well with DAK simulations, except for a few outliers. Good agreement is found between the DAK-simulated actinic flux profiles and the observations for single-layer clouds in fully overcast scenes. The instrument is suitable for operational balloon measurements because of its simplicity and low cost. It is worth further developing the instrument and launching it together with atmospheric chemistry composition sensors.

  12. [Clinico-pathological studies on the effects of pulp protection (lining) under the condition with and without smear layers].

    PubMed

    Kato, S

    1990-05-01

    Many studies have been made of pulpal reactions to pulp protection (lining) materials have always employed a smear layer between the dentin wall and the lining materials. In this study, the author has attempted to evaluate pulpal response without a smear layer and compared effects of pulp protection (lining) with and without smear layers. Materials and methods. The experiment employed 84 vital human teeth ranging in age from 14 to 54 years. The experimental teeth were divided into 4 groups for the purposes of histopathological studies and scanning electron microscope (SEM) observation. Group C: No EDTA-treatment and lining with gutta-percha temporary material, 10 cases for histopathological study and 4 cases for SEM observation. Group TC: EDTA-treatment and lining with gutta-percha temporary material, 10 cases for histopathological study and 4 cases for SEM observation. Group P: No EDTA-treatment and lining with Protect Cement. 20 cases for histopathological study and 8 cases for SEM observation. Group TP: EDTA-treatment and lining with Protect Cement. 20 cases for histopathological study and 8 cases for SEM observation. After local anesthesia, Black's simple class I cavities were made in the experimental teeth with a diamond instrument mounted on a high-speed air turbine at room temperature with a water spray coolant. In Groups TC and TP only, an aqueous solution of 0.5% EDTA (pH 7.4) 10 ml was applied from cavity floor to dentin for 60 seconds. All cavities were then washed with distilled water, and the dentin was dried with an air stream. The floor of cavities were lined with thin layers of the following materials: Group C and TC, gutta-percha temporary material (G-C Dental Industrial Co., Tokyo, Japan). Group P and TP lining of Protect Cement which is a zinc phosphate cement containing 0.5% paraformaldehyde (Neo Dental Chemical Products, Tokyo, Japan). The restoration of each cavity was then filled with silver amalgam. Experimental teeth were clinically

  13. Laser abrasion for cosmetic and medical treatment of facial actinic damage

    SciTech Connect

    David, L.M.; Lask, G.P.; Glassberg, E.; Jacoby, R.; Abergel, R.P.

    1989-06-01

    Previous studies have shown the carbon dioxide (CO/sub 2/) laser to be effective in the treatment of actinic cheilitis. After CO/sub 2/ laser abrasion, normal skin and marked cosmetic improvement of the lip were noted. In our study, twenty-three patients were treated with CO/sub 2/ laser abrasions for cosmetic improvement of facial lines and actinic changes. Pre- and postoperative histopathologic examinations were made on two patients. Preoperative examination of specimens from actinically damaged skin showed atypical keratinocytes in the basal layer of the epidermis, with overlying dense compact orthokeratosis and parakeratosis. Abundant solar elastosis was seen in the papillary dermis. Postoperative histologic specimens showed a normal-appearing epidermis with fibrosis in the papillary dermis and minimal solar elastosis (about four weeks after laser treatment). At present, various modalities are available for the regeneration of the aged skin, including chemical peels and dermabrasion. Significantly fewer complications were noted with CO/sub 2/ laser abrasion than with these methods. Thus, CO/sub 2/ laser abrasion can be useful in the cosmetic and medical treatment of the aged skin. Marked clinical and histologic improvement has been demonstrated.

  14. Coupling Between Microstrip Lines with Finite Width Ground Plane Embedded in Polyimide Layers for 3D-MMICs on Si

    NASA Technical Reports Server (NTRS)

    Ponchak, George E.; Dalton, Edan; Tentzeris, Emmanouil M.; Papapolymerou, John; Williams, W. Dan (Technical Monitor)

    2001-01-01

    Three-dimensional circuits built upon multiple layers of polyimide are required for constructing Si/SiGe monolithic microwave/millimeter-wave integrated circuits on complementary metal oxide semiconductor (CMOS) (low resistivity) Si wafers. Thin film microstrip lines (TFMS) with finite width ground planes embedded in the polyimide are often used. However, the closely spaced TFMS lines are susceptible to high levels of coupling, which degrades circuit performance. In this paper, Finite Difference Time Domain (FDTD) analysis and experimental measurements are used to show that the ground planes must be connected by via holes to reduce coupling in both the forward and backward directions.

  15. Actin Assembly at Model-Supported Lipid Bilayers

    PubMed Central

    Heath, George R.; Johnson, Benjamin R.G.; Olmsted, Peter D.; Connell, Simon D.; Evans, Stephen D.

    2013-01-01

    We report on the use of supported lipid bilayers to reveal dynamics of actin polymerization from a nonpolymerizing subphase via cationic phospholipids. Using varying fractions of charged lipid, lipid mobility, and buffer conditions, we show that dynamics at the nanoscale can be used to control the self-assembly of these structures. In the case of fluid-phase lipid bilayers, the actin adsorbs to form a uniform two-dimensional layer with complete surface coverage whereas gel-phase bilayers induce a network of randomly oriented actin filaments, of lower coverage. Reducing the pH increased the polymerization rate, the number of nucleation events, and the total coverage of actin. A model of the adsorption/diffusion process is developed to provide a description of the experimental data and shows that, in the case of fluid-phase bilayers, polymerization arises equally due to the adsorption and diffusion of surface-bound monomers and the addition of monomers directly from the solution phase. In contrast, in the case of gel-phase bilayers, polymerization is dominated by the addition of monomers from solution. In both cases, the filaments are stable for long times even when the G-actin is removed from the supernatant—making this a practical approach for creating stable lipid-actin systems via self-assembly. PMID:24268147

  16. Actin filament organization in the fish keratocyte lamellipodium

    PubMed Central

    1995-01-01

    resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton. PMID:7775574

  17. Effect of single or combined climatic and hygienic stress in four layer lines: 2. Endocrine and oxidative stress responses.

    PubMed

    Star, L; Decuypere, E; Parmentier, H K; Kemp, B

    2008-06-01

    Effects of long-term climatic stress (heat exposure), short-term hygienic stress [lipopolysaccharide (LPS)], or combined exposure to these stressors on endocrine and oxidative stress parameters of 4 layer lines (B1, WA, WB, and WF) were investigated. The lines were earlier characterized for natural humoral immune competence and survival rate. Eighty hens per line were randomly divided over 2 identical climate chambers and exposed to constant high temperature (32 degrees C) or a control temperature (21 degrees C) for 23 d. Half of the hens housed in each chamber were i.v. injected with LPS at d 1 after the start of the heat stress period. The effect of heat, LPS, or combined exposure on plasma levels of corticosterone, 3,5,3'-triiodothyronine (T(3)), glucose, uric acid (UA), and TBA reacting substances (TBARS) were investigated. Except for UA, there were no interactions between heat stress and LPS administration. Heat stress enhanced levels of corticosterone, glucose, and TBARS, whereas levels of T(3) and UA were decreased. The T(3) levels, however, were enhanced by LPS administration, whereas levels of UA were decreased. Administration of LPS had no effect on levels of corticosterone and TBARS. Because both stressors caused a reduction in feed intake, it is assumed that changes in most of the plasma levels of the endocrine and oxidative stress parameters are related with the reduction in feed intake. Neither natural humoral immune competence nor survival rate, for which the lines have been characterized, was indicative for the endocrine and oxidative stress responses to different stressors. The present data suggest that hens were able to cope with single or combined heat stress and LPS administration and that heat stress and LPS administration acted like 2 independent stressors. Furthermore, the 4 layer lines differed in response patterns and response levels; line WB was physiologically most sensitive to environmental changes.

  18. Effect of single or combined climatic and hygienic stress in four layer lines: 2. Endocrine and oxidative stress responses.

    PubMed

    Star, L; Decuypere, E; Parmentier, H K; Kemp, B

    2008-06-01

    Effects of long-term climatic stress (heat exposure), short-term hygienic stress [lipopolysaccharide (LPS)], or combined exposure to these stressors on endocrine and oxidative stress parameters of 4 layer lines (B1, WA, WB, and WF) were investigated. The lines were earlier characterized for natural humoral immune competence and survival rate. Eighty hens per line were randomly divided over 2 identical climate chambers and exposed to constant high temperature (32 degrees C) or a control temperature (21 degrees C) for 23 d. Half of the hens housed in each chamber were i.v. injected with LPS at d 1 after the start of the heat stress period. The effect of heat, LPS, or combined exposure on plasma levels of corticosterone, 3,5,3'-triiodothyronine (T(3)), glucose, uric acid (UA), and TBA reacting substances (TBARS) were investigated. Except for UA, there were no interactions between heat stress and LPS administration. Heat stress enhanced levels of corticosterone, glucose, and TBARS, whereas levels of T(3) and UA were decreased. The T(3) levels, however, were enhanced by LPS administration, whereas levels of UA were decreased. Administration of LPS had no effect on levels of corticosterone and TBARS. Because both stressors caused a reduction in feed intake, it is assumed that changes in most of the plasma levels of the endocrine and oxidative stress parameters are related with the reduction in feed intake. Neither natural humoral immune competence nor survival rate, for which the lines have been characterized, was indicative for the endocrine and oxidative stress responses to different stressors. The present data suggest that hens were able to cope with single or combined heat stress and LPS administration and that heat stress and LPS administration acted like 2 independent stressors. Furthermore, the 4 layer lines differed in response patterns and response levels; line WB was physiologically most sensitive to environmental changes. PMID:18492989

  19. Nicotiana tabacum actin-depolymerizing factor 2 is involved in actin-driven, auxin-dependent patterning.

    PubMed

    Durst, Steffen; Nick, Peter; Maisch, Jan

    2013-08-15

    Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP2) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus. PMID:23545293

  20. Actin Is Involved in Auxin-Dependent Patterning1

    PubMed Central

    Maisch, Jan; Nick, Peter

    2007-01-01

    Polar transport of auxin has been identified as a central element of pattern formation. The polarity of auxin transport is linked to the cycling of pin-formed proteins, a process that is related to actomyosin-dependent vesicle traffic. To get insight into the role of actin for auxin transport, we used patterned cell division to monitor the polarity of auxin fluxes. We show that cell division in the tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line is partially synchronized and that this synchrony can be perturbed by inhibition of auxin transport by 1-N-naphthylphthalamic acid. To address the role of actin in this synchrony, we induced a bundled configuration of actin by overexpressing mouse talin. The bundling of actin impairs the synchrony of cell division and increases the sensitivity to 1-N-naphthylphthalamic acid. Addition of the polarly transported auxins indole-3-acetic acid and 1-naphthyl acetic acid (but not 2,4-dichlorophenoxyacetic acid) restored both the normal organization of actin and the synchrony of cell division. This study suggests that auxin controls its own transport by changing the state of actin filaments. PMID:17337532

  1. Actin is involved in auxin-dependent patterning.

    PubMed

    Maisch, Jan; Nick, Peter

    2007-04-01

    Polar transport of auxin has been identified as a central element of pattern formation. The polarity of auxin transport is linked to the cycling of pin-formed proteins, a process that is related to actomyosin-dependent vesicle traffic. To get insight into the role of actin for auxin transport, we used patterned cell division to monitor the polarity of auxin fluxes. We show that cell division in the tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line is partially synchronized and that this synchrony can be perturbed by inhibition of auxin transport by 1-N-naphthylphthalamic acid. To address the role of actin in this synchrony, we induced a bundled configuration of actin by overexpressing mouse talin. The bundling of actin impairs the synchrony of cell division and increases the sensitivity to 1-N-naphthylphthalamic acid. Addition of the polarly transported auxins indole-3-acetic acid and 1-naphthyl acetic acid (but not 2,4-dichlorophenoxyacetic acid) restored both the normal organization of actin and the synchrony of cell division. This study suggests that auxin controls its own transport by changing the state of actin filaments.

  2. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

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

  3. Mechanics of Biomimetic Liposomes Encapsulating an Actin Shell.

    PubMed

    Guevorkian, Karine; Manzi, John; Pontani, Léa-Lætitia; Brochard-Wyart, Françoise; Sykes, Cécile

    2015-12-15

    Cell-shape changes are insured by a thin, dynamic, cortical layer of cytoskeleton underneath the plasma membrane. How this thin cortical structure impacts the mechanical properties of the whole cell is not fully understood. Here, we study the mechanics of liposomes or giant unilamellar vesicles, when a biomimetic actin cortex is grown at the inner layer of the lipid membrane via actin-nucleation-promoting factors. Using a hydrodynamic tube-pulling technique, we show that tube dynamics is clearly affected by the presence of an actin shell anchored to the lipid bilayer. The same force pulls much shorter tubes in the presence of the actin shell compared to bare membranes. However, in both cases, we observe that the dynamics of tube extrusion has two distinct features characteristic of viscoelastic materials: rapid elastic elongation, followed by a slower elongation phase at a constant rate. We interpret the initial elastic regime by an increase of membrane tension due to the loss of lipids into the tube. Tube length is considerably shorter for cortex liposomes at comparable pulling forces, resulting in a higher spring constant. The presence of the actin shell seems to restrict lipid mobility, as is observed in the corral effect in cells. The viscous regime for bare liposomes corresponds to a leakout of the internal liquid at constant membrane tension. The presence of the actin shell leads to a larger friction coefficient. As the tube is pulled from a patchy surface, membrane tension increases locally, leading to a Marangoni flow of lipids. As a conclusion, the presence of an actin shell is revealed by its action that alters membrane mechanics.

  4. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    NASA Technical Reports Server (NTRS)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  5. Regulation of an Actin Spring

    NASA Astrophysics Data System (ADS)

    Tam, Barney; Shin, Jennifer; Brau, Ricardo; Lang, Matthew; Mahadevan, L.; Matsudaira, Paul

    2006-03-01

    To produce motion, cells rely on the conversion of potential energy into mechanical work. One such example is the dramatic process involving the acrosome reaction of Limulus sperm, whereby a 60 μm-long bundle of actin filaments straightens from a coiled conformation to extend out of the cell in five seconds. This cellular engine and the motion it produces represent a third type of actin-based motility fundamentally different from polymerization or myosin-driven processes. The motive force for this extension originates from stored elastic energy in the overtwisted, pre-formed coil---much like a compressed mechanical spring. When the actin bundle untwists, this energy is converted to mechanical work powering the extension. We report on experiments probing the regulation of this actin spring by extracellular calcium. We find that extracellular calcium needs to be present for the spring to activate, and that calcium regulates the velocity of the extension.

  6. Chemotaxis and Actin Oscillations

    NASA Astrophysics Data System (ADS)

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

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

  7. On-line thickness measurement for two-layer systems on polymer electronic devices.

    PubMed

    Grassi, Ana Perez; Tremmel, Anton J; Koch, Alexander W; El-Khozondar, Hala J

    2013-11-18

    During the manufacturing of printed electronic circuits, different layers of coatings are applied successively on a substrate. The correct thickness of such layers is essential for guaranteeing the electronic behavior of the final product and must therefore be controlled thoroughly. This paper presents a model for measuring two-layer systems through thin film reflectometry (TFR). The model considers irregular interfaces and distortions introduced by the setup and the vertical vibration movements caused by the production process. The results show that the introduction of these latter variables is indispensable to obtain correct thickness values. The proposed approach is applied to a typical configuration of polymer electronics on transparent and non-transparent substrates. We compare our results to those obtained using a profilometer. The high degree of agreement between both measurements validates the model and suggests that the proposed measurement method can be used in industrial applications requiring fast and non-contact inspection of two-layer systems. Moreover, this approach can be used for other kinds of materials with known optical parameters.

  8. Three's company: the fission yeast actin cytoskeleton.

    PubMed

    Kovar, David R; Sirotkin, Vladimir; Lord, Matthew

    2011-03-01

    How the actin cytoskeleton assembles into different structures to drive diverse cellular processes is a fundamental cell biological question. In addition to orchestrating the appropriate combination of regulators and actin-binding proteins, different actin-based structures must insulate themselves from one another to maintain specificity within a crowded cytoplasm. Actin specification is particularly challenging in complex eukaryotes where a multitude of protein isoforms and actin structures operate within the same cell. Fission yeast Schizosaccharomyces pombe possesses a single actin isoform that functions in three distinct structures throughout the cell cycle. In this review we explore recent studies in fission yeast that help unravel how different actin structures operate in cells.

  9. Structural insights into de novo actin polymerization

    PubMed Central

    Dominguez, Roberto

    2010-01-01

    Summary Many cellular functions depend on rapid and localized actin polymerization/depolymerization. Yet, the de novo polymerization of actin in cells is kinetically unfavorable because of the instability of polymerization intermediates (small actin oligomers) and the actions of actin monomer binding proteins. Cells use filament nucleation and elongation factors to initiate and sustain polymerization. Structural biology is beginning to shed light on the diverse mechanisms by which these unrelated proteins initiate polymerization, undergo regulation, and mediate the transition of monomeric actin onto actin filaments. A prominent role is played by the W domain, which in some of these proteins occurs in tandem repeats that recruit multiple actin subunits. Pro-rich regions are also abundant and mediate the binding of profilin-actin complexes, which are the main source of polymerization competent actin in cells. Filament nucleation and elongation factors frequently interact with Rho family GTPases, which relay signals from membrane receptors to regulate actin cytoskeleton remodeling. PMID:20096561

  10. Near-infrared chemical imaging used for in-line analysis of inside adhesive layers in textile laminates.

    PubMed

    Mirschel, Gabriele; Daikos, Olesya; Scherzer, Tom; Steckert, Carsten

    2016-08-17

    This paper demonstrates for the first time that near-infrared (NIR) chemical imaging can be used for in-line analysis of textile lamination processes. In particular, it was applied for the quantitative determination of the applied coating weight and for monitoring of the spatial distribution of hot melt adhesive layers using chemometric approaches for spectra evaluation. Layers with coating weights between about 25 and 130 g m(-2) were used for the lamination of polyester fabrics and nonwovens as well as for polyurethane foam. It was shown that quantitative data with adequate precision can be actually obtained for layers applied to materials with significantly heterogeneous surface structure such as foam or for hidden layers inside fabric laminates. Even the coating weight and the homogeneity of adhesive layers in composites consisting of black textiles only could be quantitatively analyzed. The prediction errors (RMSEP) determined in an external validation of each calibration model were found to range from about 2 g m(-2) to 6 g m(-2) depending on the specific system under investigation. All calibration models were applied for chemical imaging in order to prove their performance for monitoring the thickness and the homogeneity of adhesive layers in the various textile systems. Moreover, they were used for the detection of irregularities and coating defects. Investigations were carried out with a large hyperspectral camera mounted above a conveyor. Therefore, this method allows large-area monitoring of the properties of laminar materials. Consequently, it is potentially suited for process and quality control during the lamination of fabrics, foams and other materials in field-scale. PMID:27286771

  11. An experimental study of the penetration of polycyclic aromatic hydrocarbons through a model of the bronchial lining layer

    SciTech Connect

    Gerde, P.; Scholander, P. )

    1989-04-01

    The penetration of benzo(a)pyrene (BaP) through a nonbiological experimental model of the bronchial lining layer (BLL) was studied. The purpose was to investigate how the lipid-aqueous structure of the BLL might influence the rate of penetration of polycyclic aromatic hydrocarbons (PAHs) from the ambient air to the bronchial epithelium. The experimental model was built up in a petri dish by (A) a thin layer of paraffin at the bottom, simulating the lipophilic membranes of the epithelial cells; (B) an aqueous starch gel on top of the paraffin, simulating the viscous aqueous region of the BLL; and (C) a thin layer of phosphatidylcholine, simulating the surfactant lipid layer at the air interface. BaP was administered on top of the barrier either diffusely or from a point of source, and the penetration was studied by measuring the concentration of BaP as a function of time both in the liquid phase and in the paraffin. Comparisons were made with a purely aqueous barrier without the thin phospholipid layer. The results show that the rate of penetration of BaP through the purely aqueous barrier is orders of magnitude higher than that of the lipid-aqueous barrier. A thin layer of phospholipids at the air interface thus has a tremendous influence on the rate of penetration of lipophilic substances and probably this, rather than the release rate of PAHs from their carrier particles, is the rate-determining step in the overall transport of PAHs from such particles to the bronchial epithelium.

  12. Allyl Isothiocyanate Inhibits Actin-Dependent Intracellular Transport in Arabidopsis thaliana

    PubMed Central

    Sporsheim, Bjørnar; Øverby, Anders; Bones, Atle Magnar

    2015-01-01

    Volatile allyl isothiocyanate (AITC) derives from the biodegradation of the glucosinolate sinigrin and has been associated with growth inhibition in several plants, including the model plant Arabidopsis thaliana. However, the underlying cellular mechanisms of this feature remain scarcely investigated in plants. In this study, we present evidence of an AITC-induced inhibition of actin-dependent intracellular transport in A. thaliana. A transgenic line of A. thaliana expressing yellow fluorescent protein (YFP)-tagged actin filaments was used to show attenuation of actin filament movement by AITC. This appeared gradually in a time- and dose-dependent manner and resulted in actin filaments appearing close to static. Further, we employed four transgenic lines with YFP-fusion proteins labeling the Golgi apparatus, endoplasmic reticulum (ER), vacuoles and peroxisomes to demonstrate an AITC-induced inhibition of actin-dependent intracellular transport of or, in these structures, consistent with the decline in actin filament movement. Furthermore, the morphologies of actin filaments, ER and vacuoles appeared aberrant following AITC-exposure. However, AITC-treated seedlings of all transgenic lines tested displayed morphologies and intracellular movements similar to that of the corresponding untreated and control-treated plants, following overnight incubation in an AITC-absent environment, indicating that AITC-induced decline in actin-related movements is a reversible process. These findings provide novel insights into the cellular events in plant cells following exposure to AITC, which may further expose clues to the physiological significance of the glucosinolate-myrosinase system. PMID:26690132

  13. High-Harmonic Fast-Wave Power Flow Along Magnetic Field Lines in the Scrape-Off Layer of NSTX

    SciTech Connect

    Perkins, R. J.; Hosea, J.; Kramer, G.; Ahn, Joonwook; Bell, R. E.; Diallo, A.; Gerhardt, S.; Gray, T. K.; Green, David L; Jaeger, Erwin Frederick; Jaworski, M. A.; LeBlanc, B; McLean, Adam G; Maingi, Rajesh; Phillips, C. K.; Roquemore, L.; Ryan, Philip Michael; Sabbagh, S. A.; Taylor, G.; Wilson, J. R.

    2012-01-01

    A significant fraction of high-harmonic fast-wave (HHFW) power applied to NSTX can be lost to the scrape-off layer (SOL) and deposited in bright and hot spirals on the divertor rather than in the core plasma. We show that the HHFW power flows to these spirals along magnetic field lines passing through the SOL in front of the antenna, implying that the HHFW power couples across the entire width of the SOL rather than mostly at the antenna face. This result will help guide future efforts to understand and minimize these edge losses in order to maximize fast-wave heating and current drive.

  14. High-harmonic fast-wave power flow along magnetic field lines in the scrape-off layer of NSTX.

    PubMed

    Perkins, R J; Hosea, J C; Kramer, G J; Ahn, J-W; Bell, R E; Diallo, A; Gerhardt, S; Gray, T K; Green, D L; Jaeger, E F; Jaworski, M A; LeBlanc, B P; McLean, A; Maingi, R; Phillips, C K; Roquemore, L; Ryan, P M; Sabbagh, S; Taylor, G; Wilson, J R

    2012-07-27

    A significant fraction of high-harmonic fast-wave (HHFW) power applied to NSTX can be lost to the scrape-off layer (SOL) and deposited in bright and hot spirals on the divertor rather than in the core plasma. We show that the HHFW power flows to these spirals along magnetic field lines passing through the SOL in front of the antenna, implying that the HHFW power couples across the entire width of the SOL rather than mostly at the antenna face. This result will help guide future efforts to understand and minimize these edge losses in order to maximize fast-wave heating and current drive.

  15. Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts.

    PubMed

    Chang, Wakam; Folker, Eric S; Worman, Howard J; Gundersen, Gregg G

    2013-12-01

    In migrating fibroblasts, rearward movement of the nucleus orients the centrosome toward the leading edge. Nuclear movement results from coupling rearward-moving, dorsal actin cables to the nucleus by linear arrays of nesprin-2G and SUN2, termed transmembrane actin-associated nuclear (TAN) lines. A-type lamins anchor TAN lines, prompting us to test whether emerin, a nuclear membrane protein that interacts with lamins and TAN line proteins, contributes to nuclear movement. In fibroblasts depleted of emerin, nuclei moved nondirectionally or completely failed to move. Consistent with these nuclear movement defects, dorsal actin cable flow was nondirectional in cells lacking emerin. TAN lines formed normally in cells lacking emerin and were coordinated with the erratic nuclear movements, although in 20% of the cases, TAN lines slipped over immobile nuclei. Myosin II drives actin flow, and depletion of myosin IIB, but not myosin IIA, showed similar nondirectional nuclear movement and actin flow as in emerin-depleted cells. Myosin IIB specifically coimmunoprecipitated with emerin, and emerin depletion prevented myosin IIB localization near nuclei. These results show that emerin functions with myosin IIB to polarize actin flow and nuclear movement in fibroblasts, suggesting a novel function for the nuclear envelope in organizing directional actin flow and cytoplasmic polarity.

  16. Polymerization of Actin from Maize Pollen.

    PubMed Central

    Yen, L. F.; Liu, X.; Cai, S.

    1995-01-01

    Here we describe the in vitro polymerization of actin from maize (Zea mays) pollen. The purified actin from maize pollen reported in our previous paper (X. Liu, L.F. Yen [1992] Plant Physiol 99: 1151-1155) is biologically active. In the presence of ATP, KCl, and MgCl2 the purified pollen actin polymerized into filaments. During polymerization the spectra of absorbance at 232 nm increased gradually. Polymerization of pollen actin was evidently accompanied by an increase in viscosity of the pollen actin solution. Also, the specific viscosity of pollen F-actin increased in a concentration-dependent manner. The ultraviolet difference spectrum of pollen actin is very similar to that of rabbit muscle actin. The activity of myosin ATPase from rabbit muscle was activated 7-fold by the polymerized pollen actin (F-actin). The actin filaments were visualized under the electron microscope as doubly wound strands of 7 nm diameter. If cytochalasin B was added before staining, no actin filaments were observed. When actin filaments were treated with rabbit heavy meromyosin, the actin filaments were decorated with an arrowhead structure. These results imply that there is much similarity between pollen and muscle actin. PMID:12228343

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-15

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

  19. A multigrid approach to the global stability of swept attachment-line boundary layers

    NASA Astrophysics Data System (ADS)

    Meneghello, Gianluca; Schmid, Peter; Huerre, Patrick

    2011-11-01

    A novel numerical approach to global stability analysis is presented. A multigrid framework is leveraged in order to achieve a high computational efficiency for both the base flow and eigenvalue/eigenmode computations. The base flow is obtained as the solution of the non-linear, steady-state Navier-Stokes equations at a computational cost of nearly O (n) , where n denotes the number of degrees of freedom. The eigenvectors are obtained using the same multigrid solver, coupled to an iterative shift-invert Arnoldi algorithm. The global stability analysis of the incompressible, viscous flow near the attachment line of a swept wing is then examined for a range of Reynolds numbers up to 10000. Different families of global modes are identified together with the dominant physical mechanisms underlying the perturbation dynamics in the vicinity of the swept attachment line. Financial support from Airbus and the French National Center for Scientific Research (CNRS) is gratefully acknowledged.

  20. Layers

    NASA Astrophysics Data System (ADS)

    Hong, K. J.; Jeong, T. S.; Youn, C. J.

    2014-09-01

    The temperature-dependent photoresponse characteristics of MnAl2S4 layers have been investigated, for the first time, by use of photocurrent (PC) spectroscopy. Three peaks were observed at all temperatures. The electronic origin of these peaks was associated with band-to-band transitions from the valence-band states Γ4( z), Γ5( x), and Γ5( y) to the conduction-band state Γ1( s). On the basis of the relationship between PC-peak energy and temperature, the optical band gap could be well expressed by the expression E g( T) = E g(0) - 2.80 × 10-4 T 2/(287 + T), where E g(0) was estimated to be 3.7920 eV, 3.7955 eV, and 3.8354 eV for the valence-band states Γ4( z), Γ5( x), and Γ5( y), respectively. Results from PC spectroscopy revealed the crystal-field and spin-orbit splitting were 3.5 meV and 39.9 meV. The gradual decrease of PC intensity with decreasing temperature can be explained on the basis of trapping centers associated with native defects in the MnAl2S4 layers. Plots of log J ph, the PC current density, against 1/ T, revealed a dominant trap level in the high-temperature region. By comparing PC and the Hall effect results, we confirmed that this trap level is a shallow donor 18.9 meV below the conduction band.

  1. A Replication Study for Genome-Wide Gene Expression Levels in Two Layer Lines Elucidates Differentially Expressed Genes of Pathways Involved in Bone Remodeling and Immune Responsiveness

    PubMed Central

    Habig, Christin; Geffers, Robert; Distl, Ottmar

    2014-01-01

    The current replication study confirmed significant differences in gene expression profiles of the cerebrum among the two commercial layer lines Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB). Microarray analyses were performed for 30 LSL and another 30 LB laying hens kept in the small group housing system Eurovent German. A total of 14,103 microarray probe sets using customized Affymetrix ChiGene-1_0-st Arrays with 20,399 probe sets were differentially expressed among the two layer lines LSL and LB (FDR adjusted P-value <0.05). An at least 2-fold change in expression levels could be observed for 388 of these probe sets. In LSL, 214 of the 388 probe sets were down- and 174 were up-regulated and vice versa for the LB layer line. Among the 174 up-regulated probe sets in LSL, we identified 51 significantly enriched Gene ontology (GO) terms of the biological process category. A total of 63 enriched GO-terms could be identified for the 214 down-regulated probe sets of the layer line LSL. We identified nine genes significantly differentially expressed between the two layer lines in both microarray experiments. These genes play a crucial role in protection of neuronal cells from oxidative stress, bone mineral density and immune response among the two layer lines LSL and LB. Thus, the different regulation of these genes may significantly contribute to phenotypic trait differences among these layer lines. In conclusion, these novel findings provide a basis for further research to improve animal welfare in laying hens and these layer lines may be of general interest as an animal model. PMID:24922511

  2. Lines

    ERIC Educational Resources Information Center

    Mires, Peter B.

    2006-01-01

    National Geography Standards for the middle school years generally stress the teaching of latitude and longitude. There are many creative ways to explain the great grid that encircles our planet, but the author has found that students in his college-level geography courses especially enjoy human-interest stories associated with lines of latitude…

  3. 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.

  4. A large mobility of hydrophilic molecules at the outmost layer controls the protein adsorption and adhering behavior with the actin fiber orientation of human umbilical vein endothelial cells (HUVEC).

    PubMed

    Kakinoki, Sachiro; Seo, Ji-Hun; Inoue, Yuuki; Ishihara, Kazuhiko; Yui, Nobuhiko; Yamaoka, Tetsuji

    2013-01-01

    Adhesion behaviors of human umbilical vein endothelial cells (HUVECs) are interestingly affected by the mobility of hydrophilic chains on the material surfaces. Surfaces with different molecular mobilities were prepared using ABA-type block copolymers consisting polyrotaxane (PRX) or poly(ethylene glycol) (PEG) central block (A block), and amphiphilic anchoring B blocks of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). Two different molecular mobilities of the PRX chains were designed by using normal α-cyclodextrin (α-CD) or α-CD whose hydroxyl groups were converted to methoxy groups in a given ratio to improve its molecular mobility (PRX-PMB and OMe-PRX-PMB). The surface mobility of these materials was assessed as the mobility factor (Mf), which is measured by quartz crystal microbalance with dissipation monitoring system. HUVECs adhered on OMe-PRX-PMB surface much more than PRX-PMB and PMB-block-PEG-block-PMB (PEG-PMB) surfaces. These different HUVEC adhesions were correlated with the density of cell-binding site of adsorbed fibronectin. In addition, the alignment of the actin cytoskeleton of adhered HUVECs was strongly suppressed on the PEG-PMB, PRX-PMB, and OMe-PRX-PMB in response to the increased Mf value. Remarkably, the HUVECs adhered on the OMe-PRX-PMB surface with much less actin organization. We concluded that not only the cell adhesion but also the cellular function are regulated by the molecular mobility of the outmost material surfaces. PMID:23796033

  5. Actin polymerization and intracellular solvent flow in cell surface blebbing

    PubMed Central

    1995-01-01

    The cortical actin gel of eukaryotic cells is postulated to control cell surface activity. One type of protrusion that may offer clues to this regulation are the spherical aneurysms of the surface membrane known as blebs. Blebs occur normally in cells during spreading and alternate with other protrusions, such as ruffles, suggesting similar protrusive machinery is involved. We recently reported that human melanoma cell lines deficient in the actin filament cross-linking protein, ABP-280, show prolonged blebbing, thus allowing close study of blebs and their dynamics. Blebs expand at different rates of volume increase that directly predict the final size achieved by each bleb. These rates decrease as the F-actin concentration of the cells increase over time after plating on a surface, but do so at lower concentrations in ABP-280 expressing cells. Fluorescently labeled actin and phalloidin injections of blebbing cells indicate that a polymerized actin structure is not present initially, but appears later and is responsible for stopping further bleb expansion. Therefore, it is postulated that blebs occur when the fluid-driven expansion of the cell membrane is sufficiently rapid to initially outpace the local rate of actin polymerization. In this model, the rate of intracellular solvent flow driving this expansion decreases as cortical gelation is achieved, whether by factors such as ABP-280, or by concentrated actin polymers alone, thereby leading to decreased size and occurrence of blebs. Since the forces driving bleb extension would always be present in a cell, this process may influence other cell protrusions as well. PMID:7790356

  6. State transitions of actin cortices in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Tan, Tzer Han; Keren, Kinneret; Mackintosh, Fred; Schmidt, Christoph; Fakhri, Nikta

    Most animal cells are enveloped by a thin layer of actin cortex which governs the cell mechanics. A functional cortex must be rigid to provide mechanical support while being flexible to allow for rapid restructuring events such as cell division. To satisfy these requirements, the actin cortex is highly dynamic with fast actin turnover and myosin-driven contractility. The regulatory mechanism responsible for the transition between a mechanically stable state and a restructuring state is not well understood. Here, we develop a technique to map the dynamics of reconstituted actin cortices in emulsion droplets using IR fluorescent single-walled carbon nanotubes (SWNTs). By increasing crosslinker concentration, we find that a homogeneous cortex transitions to an intermediate state with broken rotational symmetry and a globally contractile state which further breaks translational symmetry. We apply this new dynamic mapping technique to cortices of live starfish oocytes in various developmental stages. To identify the regulatory mechanism for steady state transitions, we subject the oocytes to actin and myosin disrupting drugs.

  7. Rheology-modulated contact line dynamics of an immiscible binary system under electrical double layer phenomena.

    PubMed

    Mondal, Pranab Kumar; DasGupta, Debabrata; Chakraborty, Suman

    2015-09-01

    We investigate the electrically driven contact line dynamics of a binary fluid system constituted by one Newtonian and another non-Newtonian fluid in a narrow fluidic channel with chemically patched walls. We use a power-law model to describe the rheology of the non-Newtonian fluid and a diffuse interface phase-field method to model the dynamics of multiple phases. We bring out the alteration in the interfacial dynamics as attributable to the rheology-driven modifications in the interfacial stress and its interplay with the Maxwell stress originating from electrokinetic effects. PMID:26221770

  8. Ion diode performance on a positive polarity inductive voltage adder with layered magnetically insulated transmission line flow

    SciTech Connect

    Hinshelwood, D. D.; Schumer, J. W.; Allen, R. J.; Commisso, R. J.; Jackson, S. L.; Murphy, D. P.; Phipps, D.; Swanekamp, S. B.; Weber, B. V.; Ottinger, P. F.; Apruzese, J. P.; Cooperstein, G.; Young, F. C.

    2011-05-15

    A pinch-reflex ion diode is fielded on the pulsed-power machine Mercury (R. J. Allen, et al., 15th IEEE Intl. Pulsed Power Conf., Monterey, CA, 2005, p. 339), which has an inductive voltage adder (IVA) architecture and a magnetically insulated transmission line (MITL). Mercury is operated in positive polarity resulting in layered MITL flow as emitted electrons are born at a different potential in each of the adder cavities. The usual method for estimating the voltage by measuring the bound current in the cathode and anode of the MITL is not accurate with layered flow, and the interaction of the MITL flow with a pinched-beam ion diode load has not been studied previously. Other methods for determining the diode voltage are applied, ion diode performance is experimentally characterized and evaluated, and circuit and particle-in-cell (PIC) simulations are performed. Results indicate that the ion diode couples efficiently to the machine operating at a diode voltage of about 3.5 MV and a total current of about 325 kA, with an ion current of about 70 kA of which about 60 kA is proton current. It is also found that the layered flow impedance of the MITL is about half the vacuum impedance.

  9. Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

    PubMed

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R; Bryce, Nicole S; Whan, Renee M; Hardeman, Edna C; Fath, Thomas; Schevzov, Galina; Gunning, Peter W

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  11. The use of human amniotic fluid mesenchymal stem cells as the feeder layer to establish human embryonic stem cell lines.

    PubMed

    Soong, Yung-Kwei; Huang, Shang-Yu; Yeh, Chiu-Hsiang; Wang, Tzu-Hao; Chang, Kuo-Hsuan; Cheng, Po-Jen; Shaw, S W Steven

    2015-12-01

    Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into the three germ layers and possibly all tissues of the human body. To fulfil the clinical potentials for cell-based therapy, banks of hESC lines that express different combinations of the major histocompatibility genes should be established, preferably without exposing such cells to animal cells and proteins. In this study, we tested human amniotic fluid mesenchymal stem cells (AFMSCs) as feeder cells to support the growth of hESCs. Our results indicated that mitomycin-treated AFMSCs were able to support the newly established hESC lines CGLK-1 and CGLK-2. The hESC colonies cultured on AFMSCs expressed alkaline phosphatase (ALK-P), SSEA-4, TRA-1-60, TRA-1-81, Oct-4, Nanog and Sox-2, which are markers for undifferentiated hESCs. Chromosomal analyses of both hESC lines, CGLK-1 and CGLK-2, which were cultured on AFMSC feeders for 22 and 14 passages, respectively, were confirmed to be normal karyotypes (46, XX). The ability of AFMSCs as feeder cells to maintain the undifferentiated growth and pluripotency of hESCs was confirmed by in vivo formation of teratomas derived on AFMSC hESCs in severe combined immune-compromised mice. The use of AFMSCs for feeder cells to culture hESCs has several advantages, in that AFMSCs are not tumourigenic and can be expanded extensively with a short doubling time.

  12. How two-dimensional brick layer J-aggregates differ from linear ones: Excitonic properties and line broadening mechanisms

    NASA Astrophysics Data System (ADS)

    Dijkstra, Arend G.; Duan, Hong-Guang; Knoester, Jasper; Nelson, Keith A.; Cao, Jianshu

    2016-04-01

    We study the excitonic coupling and homogeneous spectral line width of brick layer J-aggregate films. We begin by analysing the structural information revealed by the two-exciton states probed in two-dimensional spectra. Our first main result is that the relation between the excitonic couplings and the spectral shift in a two-dimensional structure is different (larger shift for the same nearest neighbour coupling) from that in a one-dimensional structure, which leads to an estimation of dipolar coupling in two-dimensional lattices. We next investigate the mechanisms of homogeneous broadening—population relaxation and pure dephasing—and evaluate their relative importance in linear and two-dimensional aggregates. Our second main result is that pure dephasing dominates the line width in two-dimensional systems up to a crossover temperature, which explains the linear temperature dependence of the homogeneous line width. This is directly related to the decreased density of states at the band edge when compared with linear aggregates, thus reducing the contribution of population relaxation to dephasing. Pump-probe experiments are suggested to directly measure the lifetime of the bright state and can therefore support the proposed model.

  13. Bacterial Actins? An Evolutionary Perspective

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.; York, Amanda L.

    2003-01-01

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

  14. A Robust Actin Filaments Image Analysis Framework

    PubMed Central

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-01-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a ‘cartoon’ part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the ‘cartoon’ image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts

  15. A Robust Actin Filaments Image Analysis Framework.

    PubMed

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-08-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a 'cartoon' part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the 'cartoon' image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts grown in

  16. Grouping Annotations on the Subcellular Layered Interactome Demonstrates Enhanced Autophagy Activity in a Recurrent Experimental Autoimmune Uveitis T Cell Line

    PubMed Central

    Zhao, Yu; Dong, Yucui; Ju, Huanyu; Yang, Jinfeng; Sun, Jianhua; Li, Xia; Ren, Huan

    2014-01-01

    Human uveitis is a type of T cell-mediated autoimmune disease that often shows relapse–remitting courses affecting multiple biological processes. As a cytoplasmic process, autophagy has been seen as an adaptive response to cell death and survival, yet the link between autophagy and T cell-mediated autoimmunity is not certain. In this study, based on the differentially expressed genes (GSE19652) between the recurrent versus monophasic T cell lines, whose adoptive transfer to susceptible animals may result in respective recurrent or monophasic uveitis, we proposed grouping annotations on a subcellular layered interactome framework to analyze the specific bioprocesses that are linked to the recurrence of T cell autoimmunity. That is, the subcellular layered interactome was established by the Cytoscape and Cerebral plugin based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. The analysis showed that significant bioprocesses with autophagy were orchestrated in the cytoplasmic layered interactome and that mTOR may have a regulatory role in it. Furthermore, by setting up recurrent and monophasic uveitis in Lewis rats, we confirmed by transmission electron microscopy that, in comparison to the monophasic disease, recurrent uveitis in vivo showed significantly increased autophagy activity and extended lymphocyte infiltration to the affected retina. In summary, our framework methodology is a useful tool to disclose specific bioprocesses and molecular targets that can be attributed to a certain disease. Our results indicated that targeted inhibition of autophagy pathways may perturb the recurrence of uveitis. PMID:25116327

  17. Vortex lines in layered superconductors. I. From 3D to 2D behaviour

    NASA Astrophysics Data System (ADS)

    Feinberg, D.

    1994-02-01

    The fundamental aspects of vortices in layered superconductors (natural or artificial multilayered materials) are reviewed, focusing on the role of anisotropy and very short coherence lengths. These materials divide into three classes, with increasing T_c's : chalcogenides, organic superconductors and high-T_c copper oxides. The first part of the paper summarizes the quantitative features of the vortex lattice, due to the incorporation of anisotropy in the 3D Ginzburg-Landau or London descriptions : anisotropy of critical fields and vortex lattice, elastic coefficients and melting. This kind of model describes most of the properties of moderately anisotropic compounds as Y : 123. The second part concerns the Josephson-coupled layered systems and identifies in which regimes vortices exhibit a quasi-2D character. Qualitatively new features as Josephson vortices, 2D vortices, Kosterlitz-Thouless transition and lock-in of vortices are reviewed. This analysis is adapted to compounds as Bi : 2212 or multilayers, but also to Y : 123 for some aspects. On passe en revue les aspects fondamentaux des vortex dans les supraconducteurs lamellaires (naturels ou superréseaux artificiels), en mettant l'accent sur le rôle de l'anisotropie et des très courtes longueurs de cohérence. Ces composés se divisent en trois classes, de T_c croissants : chalcogénures, supraconducteurs organiques et oxydes de cuivre à haut T_c. La première partie de l'article résume les aspects quantitatifs dus à l'incorporation de l'anisotropie dans les descriptions 3D Ginzburg-Landau ou London du réseau de vortex.: anisotropie des champs critiques et du réseau de vortex, coefficients élastiques et fusion. Ce type de modèle décrit une grande partie des propriétés des composés modérément anisotropes tels que Y : 123. La seconde partie concerne les systèmes lamellaires à couplage Josephson et identifie dans quels régimes les vortex présentent un caractère quasi-2D. Des effets

  18. Fascin regulates nuclear actin during Drosophila oogenesis.

    PubMed

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

    2016-10-01

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

  19. Fascin regulates nuclear actin during Drosophila oogenesis.

    PubMed

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

    2016-10-01

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

  20. O3-induced formation of bioactive lipids: estimated surface concentrations and lining layer effects.

    PubMed

    Postlethwait, E M; Cueto, R; Velsor, L W; Pryor, W A

    1998-06-01

    Recent evidence suggests that inhaled ozone (O3) does not induce toxicity via direct epithelial interactions. Reactions with epithelial lining fluid (ELF) constituents limit cellular contact and generate products, including lipid ozonation products, postulated to initiate pathophysiological cascades. To delineate specific aspects of lipid ozonation product formation and to estimate in situ surface concentrations, we studied the O3 absorption characteristics of ELF constituent mixtures and measured hexanal, heptanal, and nonanal yields as a function of ascorbic acid (AH2) concentration. Exposures of isolated rat lungs, bronchoalveolar lavage fluid (BALF) and egg phosphatidylcholine (PC) liposomes were conducted. 1) O3 absorption by AH2, uric acid, and albumin exceeded that by egg PC and glutathione. O3 reaction with egg PC occurred when AH2 concentrations were reduced. 2) Aldehydes were produced in low yield during lung and BALF exposures in a time- and O3 concentration-dependent manner. 3) Diminishing BALF AH2 content lowered O3 uptake but increased aldehyde yields. Conversely, AH2 addition to egg PC increased O3 uptake but reduced aldehyde yields. Estimations of bioactive ozonation and autoxidation product accumulation within the ELF suggested possible nanomolar to low micromolar concentrations. The use of reaction products as metrics of O3 exposure may have intrinsic sensitivity and specificity limitations. Moreover, due to the heterogenous nature of O3 reactions within the ELF, dose-response relationships may not be linear with respect to O3 absorption.

  1. The reconstitution of actin polymerization on liposomes.

    PubMed

    Stamnes, Mark; Xu, Weidong

    2010-01-01

    Membrane-associated actin polymerization is of considerable interest due to its role in cell migration and the motility of intracellular organelles. Intensive research efforts are underway to investigate the physiological role of membrane-associated actin as well as the regulation and mechanics of actin assembly. Branched actin polymerization on membranes is catalyzed by the Arp2/3 complex. Signaling events leading to the activation of the guanosine triphosphate (GTP)-binding protein Cdc42 stimulate Arp2/3-dependent actin polymerization. We have studied the role of Cdc42 at the Golgi apparatus in part by reconstituting actin polymerization on isolated Golgi membranes and on liposomes. In this manner, we showed that cytosolic proteins are sufficient for actin assembly on a phospholipid bilayer. Here we describe methods for the cell-free reconstitution of membrane-associated actin polymerization using liposomes and brain cytosol.

  2. Measurements of Spatial Line Emission Profiles in the Main Scrape-Off Layer of the DIII-D Tokamak

    SciTech Connect

    Groth, M; Ellis, R; Brooks, N; Fenstermacher, M; Lasnier, C; Meyer, W; Moller, J

    2009-06-05

    A video camera system is described that measures the spatial distribution of visible line emission emitted from the main scrape-off layer (SOL) of plasmas in the DIII-D tokamak. A wide-angle lens installed on an equatorial port and an in-vessel mirror which intercepts part of the lens view provide simultaneous tangential views of the SOL on the low-field and high-field sides of the plasma's equatorial plane. Tomographic reconstruction techniques are used to calculate the 2-D poloidal profiles from the raw data, and 1-D poloidal profiles simulating chordal views of other optical diagnostics from the 2-D profiles. The 2-D profiles can be compared with SOL plasma simulations; the 1-D profiles with measurements from spectroscopic diagnostics. Sample results are presented which elucidate carbon transport in plasmas with toroidally uniform injection of methane and argon transport in disruption mitigation experiments with massive gas jet injection.

  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. Actin in hair cells and hearing loss.

    PubMed

    Drummond, Meghan C; Belyantseva, Inna A; Friderici, Karen H; Friedman, Thomas B

    2012-06-01

    Hereditary deafness is genetically heterogeneous such that mutations of many different genes can cause hearing loss. This review focuses on the evidence and implications that several of these deafness genes encode actin-interacting proteins or actin itself. There is a growing appreciation of the contribution of the actin interactome in stereocilia development, maintenance, mechanotransduction and malfunction of the auditory system.

  5. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization

    PubMed Central

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2016-01-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis. PMID:25664724

  6. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.

    PubMed

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2015-03-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis.

  7. Effect of single or combined climatic and hygienic stress on natural and specific humoral immune competence in four layer lines.

    PubMed

    Star, L; Nieuwland, M G B; Kemp, B; Parmentier, H K

    2007-09-01

    Effects of long-term climatic stress (heat exposure), short-term hygienic stress [lipopolysaccharide (LPS)], or a combination of both challenges on the immune competence of 4 layer lines was investigated. The lines were earlier characterized for natural humoral immune competence and survival rate. Eighty hens per line were randomly divided over 2 identical climate chambers and exposed to a constant high temperature (32 degrees C) or a control temperature (21 degrees C) for 23 d. Half of the hens housed in each chamber were i.v. injected with LPS at d 1 after the start of the heat stress period. Within each of the treatment groups, half of the hens were s.c. immunized with human serum albumin (HuSA) at d 2 after the start of the heat stress period to measure specific antibody (Ab) titers to HuSA. The effect of heat, LPS, or a combined challenge on specific Ab titers to HuSA, natural Ab titers to keyhole limpet hemocyanin or HuSA (in hens that were not immunized with HuSA), and activity of the classical and alternative complement pathways were investigated. Heat stress enhanced specific and natural immune responses. Administration of LPS enhanced natural immune responses but decreased specific immune responses. The lack of interaction between heat stress and LPS administration, except for natural Ab titers to HuSA, suggest that these were 2 independent stressors. The lines had a similar response pattern but differed in the response level. Neither natural humoral immune competence nor survival rate, for which the lines had been characterized, was indicative of the specific and natural immune response to different stressors. Lipopolysaccharide and heat stress initiated sequential responses over time, with an earlier effect of short-term LPS exposure (within the first and second week) and a later effect of long-term heat exposure (within the second and third week). These data suggest that LPS and heat stress affect the natural and specific immune competence of laying

  8. Effect of resin-modified glass-ionomer cement lining and composite layering technique on the adhesive interface of lateral wall

    PubMed Central

    AZEVEDO, Larissa Marinho; CASAS-APAYCO, Leslie Carol; VILLAVICENCIO ESPINOZA, Carlos Andres; WANG, Linda; NAVARRO, Maria Fidela de Lima; ATTA, Maria Teresa

    2015-01-01

    Interface integrity can be maintained by setting the composite in a layering technique and using liners. Objective The aim of this in vitro study was to verify the effect of resin-modified glass-ionomer cement (RMGIC) lining and composite layering technique on the bond strength of the dentin/resin adhesive interface of lateral walls of occlusal restorations. Material and Methods Occlusal cavities were prepared in 52 extracted sound human molars, randomly assigned into 4 groups: Group 2H (control) – no lining + two horizontal layers; Group 4O: no lining + four oblique layers; Group V-2H: RMGIC lining (Vitrebond) + two horizontal layers; and Group V-4O: RMGIC lining (Vitrebond) + four oblique layers. Resin composite (Filtek Z250, 3M ESPE) was placed after application of an adhesive system (Adper™ Single Bond 2, 3M ESPE) dyed with a fluorescent reagent (Rhodamine B) to allow confocal microscopy analysis. The teeth were stored in deionized water at 37oC for 24 hours before being sectioned into 0.8 mm slices. One slice of each tooth was randomly selected for Confocal Laser Scanning Microscopy (CLSM) analysis. The other slices were sectioned into 0.8 mm x 0.8 mm sticks to microtensile bond strength test (MPa). Data were analyzed by two-way ANOVA and Fisher’s test. Results There was no statistical difference on bond strength among groups (p>0.05). CLSM analysis showed no significant statistical difference regarding the presence of gap at the interface dentin/resin among groups. Conclusions RMGIC lining and composite layering techniques showed no effect on the microtensile bond strength and gap formation at the adhesive interface of lateral walls of high C-factor occlusal restorations. PMID:26221927

  9. Changes in actin dynamics are involved in salicylic acid signaling pathway.

    PubMed

    Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

    2014-06-01

    Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented.

  10. Mechanism of Actin-Based Motility

    NASA Astrophysics Data System (ADS)

    Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

    2001-05-01

    Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

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

    PubMed

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

    2011-10-01

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

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

    PubMed

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

    1993-07-01

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

  13. The actin of muscle and fibroblasts.

    PubMed Central

    Anderson, P J

    1976-01-01

    The isolation and quantification of an 18-residue peptide from the N-terminal region of chicken actin was used to quantify the amount of actin in acetone-dried powders of chicken breast muscle and chicken-embryo fibroblasts. Either isotope dilution or double labelling can be used for peptide quantification. About 17% of the protein of chicken breast muscle was estimated to be actin. However, only 0.25% of the protein of chicken-embryo fibroblasts was determined to be actin by quantification of this peptide. The actin content of fibroblasts may be low or the amino acid sequences of muscle and fibroblast actin may differ in the N-terminal region. The methodology used can be extended to examine whether other regions of muscle actin sequence are present in fibroblasts or other cell types. PMID:938480

  14. Quantifying actin wave modulation on periodic topography

    NASA Astrophysics Data System (ADS)

    Guven, Can; Driscoll, Meghan; Sun, Xiaoyu; Parker, Joshua; Fourkas, John; Carlsson, Anders; Losert, Wolfgang

    2014-03-01

    Actin is the essential builder of the cell cytoskeleton, whose dynamics are responsible for generating the necessary forces for the formation of protrusions. By exposing amoeboid cells to periodic topographical cues, we show that actin can be directionally guided via inducing preferential polymerization waves. To quantify the dynamics of these actin waves and their interaction with the substrate, we modify a technique from computer vision called ``optical flow.'' We obtain vectors that represent the apparent actin flow and cluster these vectors to obtain patches of newly polymerized actin, which represent actin waves. Using this technique, we compare experimental results, including speed distribution of waves and distance from the wave centroid to the closest ridge, with actin polymerization simulations. We hypothesize the modulation of the activity of nucleation promotion factors on ridges (elevated regions of the surface) as a potential mechanism for the wave-substrate coupling. Funded by NIH grant R01GM085574.

  15. Physical processes in an electron current layer causing intense plasma heating and formation of x-lines

    NASA Astrophysics Data System (ADS)

    Singh, Nagendra; Khazanov, Igor; Wells, B. E.

    2015-05-01

    We study the evolution of an electron current layer (ECL) through its several stages by means of three-dimensional particle-in-cell (PIC) simulations with ion to electron mass ratio M/me = 400. An ECL evolves through the following stages: (i) Electrostatic (ES) current-driven instability (CDI) soon after its formation with half width w about 2 electron skin depth (de), (ii) current disruption in the central part of the ECL by trapping of electrons and generation of anomalous resistivity, (iii) electron tearing instability (ETI) with significantly large growth rates in the lower end of the whistler frequency range, (iv) widening of the ECL and modulation of its width by the ETI, (v) gradual heating of electrons by the CDI-driven ES ion modes create the condition that the electrons become hotter than the ions, (vi) despite the reduced electron drift associated with the current disruption by the CDI, the enhanced electron temperature continues to favor a slow growth of the ion waves reaching nonlinear amplitudes, (vii) the nonlinear ion waves undergo modulation and collapse into localized density cavities containing spiky electric fields like in double layers (DLs), (viii) such spiky electric fields are very effective in further rapid heating of both electrons and ions. As predicted by the electron magnetohydrodynamic (EMHD) theories, the ETI growth rate maximizes at wave numbers in the range 0.4 < kxW < 0.8 where kx is the wave number parallel to the ECL magnetic field and w is the evolving half width of the ECL. The developing ETI generates in-plane currents that support out-of-plane magnetic fields around the emerging x-lines. The ETI and the spiky electrostatic structures are accompanied by fluctuations in the magnetic fields near and above the lower-hybrid (ion plasma) frequency, including the whistler frequency range. We compare our results with experimental results and satellite observation.

  16. Photochemical ozone production in tropical squall line convection during NASA Global Tropospheric Experiment/Amazon Boundary Layer Experiment 2A

    NASA Technical Reports Server (NTRS)

    Pickering, Kenneth E.; Thompson, Anne M.; Tao, Wei-Kuo; Simpson, Joanne; Scala, John R.

    1991-01-01

    The role of convection was examined in trace gas transport and ozone production in a tropical dry season squall line sampled on August 3, 1985, during NASA Global Tropospheric Experiment/Amazon Boundary Layer Experiment 2A (NASA GTE/ABLE 2A) in Amazonia, Brazil. Two types of analyses were performed. Transient effects within the cloud are examined with a combination of two-dimensional cloud and one-dimensional photochemical modeling. Tracer analyses using the cloud model wind fields yield a series of cross sections of NO(x), CO, and O3 distribution during the lifetime of the cloud; these fields are used in the photochemical model to compute the net rate of O3 production. At noon, when the cloud was mature, the instantaneous ozone production potential in the cloud is between 50 and 60 percent less than in no-cloud conditions due to reduced photolysis and cloud scavenging of radicals. Analysis of cloud inflows and outflows is used to differentiate between air that is undisturbed and air that has been modified by the storm. These profiles are used in the photochemical model to examine the aftereffects of convective redistribution in the 24-hour period following the storm. Total tropospheric column O3 production changed little due to convection because so little NO(x) was available in the lower troposphere. However, the integrated O3 production potential in the 5- to 13-km layer changed from net destruction to net production as a result of the convection. The conditions of the August 3, 1985, event may be typical of the early part of the dry season in Amazonia, when only minimal amounts of pollution from biomass burning have been transported into the region.

  17. Evidence for a species of nuclear actin distinct from cytoplasmic and muscles actins.

    PubMed

    Bremer, J W; Busch, H; Yeoman, L C

    1981-03-31

    Nuclear actin (protein BJ) has been isolated from the chromatin of Novikoff hepatoma ascites cells and purified to homogeneity by selective extraction, Sepharose CL-6B chromatography, and preparative polyacrylamide gel electrophoresis. A comparison of nuclear and cytoplasmic actins from Novikoff hepatoma cells and rabbit muscle actin was made by amino acid analysis, isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional peptide mapping procedures. By these criteria, all of the proteins compared are actins, but each is chemically distinct. It was concluded, therefore, that nuclear actin is similar to, but not identical with, cytoplasmic actin isolated from Novikoff hepatoma cells. A striking similarity in peptide charge and migration as shown by peptide map analysis was observed for nuclear and rabbit skeletal muscle actins. This may indicate that nuclear actin has the capacity for contractile function. In addition, the actins synthesized in Novikoff hepatoma cells may results from more than two structural genes.

  18. Rapid actin-dependent viral motility in live cells.

    PubMed

    Vaughan, Joshua C; Brandenburg, Boerries; Hogle, James M; Zhuang, Xiaowei

    2009-09-16

    During the course of an infection, viruses take advantage of a variety of mechanisms to travel in cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. To study viral motility within the intrinsically heterogeneous environment of the cell, we have developed a motility assay that allows for the global and unbiased analysis of tens of thousands of virus trajectories in live cells. Using this assay, we discovered that poliovirus exhibits anomalously rapid intracellular movement that was independent of microtubules, a common track for fast and directed cargo transport. Such rapid motion, with speeds of up to 5 microm/s, allows the virus particles to quickly explore all regions of the cell with the exception of the nucleus. The rapid, microtubule-independent movement of poliovirus was observed in multiple human-derived cell lines, but appeared to be cargo-specific. Other cargo, including a closely related picornavirus, did not exhibit similar motility. Furthermore, the motility is energy-dependent and requires an intact actin cytoskeleton, suggesting an active transport mechanism. The speed of this microtubule-independent but actin-dependent movement is nearly an order of magnitude faster than the fastest speeds reported for actin-dependent transport in animal cells, either by actin polymerization or by myosin motor proteins.

  19. Structural Characterization of the Binding of Myosin*ADP*Pi to Actin in Permeabilized Rabbit Psoas Muscle

    SciTech Connect

    Xu,S.; Gu, J.; Belknap, B.; White, H.; Yu, L.

    2006-01-01

    the absence of PEG. When the binding between actin and myosin was increased, both the myosin layer lines and the actin layer lines increased in intensity, but the intensity profiles did not change. The configuration (mode) of attachment in the A{center_dot}M{center_dot}ADP{center_dot}P{sub i} state is thus unique among the intermediate attached states of the cross-bridge ATP hydrolysis cycle. One of the simplest explanations is that both myosin filaments and actin filaments are stabilized (e.g., undergo reduced spatial fluctuations) by the attachment. The alignment of the myosin heads in the thick filaments and the alignment of the actin monomers in the thin filaments are improved as a result. The compact atomic structure of M{center_dot}ADP{center_dot}P{sub i} with strongly coupled domains may contribute to the unique attachment configuration: the 'primed' myosin heads may function as 'transient struts' when attached to the thin filaments.

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

    PubMed

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

    2015-08-01

    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.

  1. Evaluation of bone strength, keel bone status, plumage condition and egg quality of two layer lines kept in small group housing systems.

    PubMed

    Habig, C; Distl, O

    2013-01-01

    1. The aim of the present study was to determine the influence of large (54 or 60) and small (36 or 40) group sizes and tiers of the small group housing system "Eurovent German" on tibia and humerus bone breaking strength, keel bone status, plumage condition and egg quality for two commercial layer lines, Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB), at the same stocking density (890 or 830 cm(2)/bird). 2. In 4 consecutive trials, 4752 hens were recorded for keel bone status. Evaluation of plumage condition was made for 1440 hens and bone breaking strength was recorded for 1200 hens. A total of 4962 eggs were analysed for internal and external egg quality traits. Analyses involved 30 small group compartments per trial. 3. The layer line had a much more pronounced influence on humerus breaking strength than on tibia breaking strength. 4. Plumage condition, particularly on the neck, was positively correlated with humerus breaking strength in both layer lines. 5. An average of 34% of LB and 23% of LSL hens showed keel bone deformities, with higher proportions of slight deformities. 6. Slight keel bone deformities, rather than moderate to severe, increased significantly during the laying period. 7. Stocking density had no influence on bone breaking strength, keel bone status and egg quality traits. 8. LB layers had a 1.4-fold higher humerus, but only a 1.06-fold higher tibia breaking strength compared to LSL layers. 9. Tibia breaking strength was significantly affected by the interaction of group size and layer line. LSL layers in small groups had lower tibia breaking strengths than those of the large groups.

  2. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

    Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder. PMID:241335

  3. Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening.

    PubMed

    Gentry, Brian S; van der Meulen, Stef; Noguera, Philippe; Alonso-Latorre, Baldomero; Plastino, Julie; Koenderink, Gijsje H

    2012-11-01

    Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.

  4. Effects of the F-actin inhibitor latrunculin A on the budding yeast Saccharomyces cerevisiae.

    PubMed

    Kopecká, Marie; Yamaguchi, Masashi; Kawamoto, Susumu

    2015-07-01

    Our basic cell biology research was aimed at investigating the effect on eukaryotic cells of the sudden loss of the F-actin cytoskeleton. Cells treated with latrunculin A (LA) in yeast extract peptone dextrose (YEPD) medium were examined using phase-contrast and fluorescent microscopy, freeze-substitution, transmission and scanning electron microscopy, counted using a Bürker chamber and their absorbance measured. The cells responded to the presence of LA, an F-actin inhibitor, with the disappearance of actin patches, actin cables and actin rings. This resulted in the formation of larger spherical cells with irregular morphology in the cell walls and ultrastructural disorder of the cell organelles and secretory vesicles. Instead of buds, LA-inhibited cells formed only 'table-mountain-like' wide flattened swellings without apical growth with a thinner glucan cell-wall layer containing β-1,3-glucan microfibrils. The LA-inhibited cells lysed. Actin cables and patches were required for bud formation and bud growth. In addition, actin patches were required for the formation of β-1,3-glucan microfibrils in the bud cell wall. LA has fungistatic, fungicidal and fungilytic effects on the budding yeast Saccharomyces cerevisiae.

  5. The actin cytoskeleton in endothelial cell phenotypes

    PubMed Central

    Prasain, Nutan; Stevens, Troy

    2009-01-01

    Endothelium forms a semi-permeable barrier that separates blood from the underlying tissue. Barrier function is largely determined by cell-cell and cell-matrix adhesions that define the limits of cell borders. Yet, such cell-cell and cell-matrix tethering is critically reliant upon the nature of adherence within the cell itself. Indeed, the actin cytoskeleton fulfills this essential function, to provide a strong, dynamic intracellular scaffold that organizes integral membrane proteins with the cell’s interior, and responds to environmental cues to orchestrate appropriate cell shape. The actin cytoskeleton is comprised of three distinct, but interrelated structures, including actin cross-linking of spectrin within the membrane skeleton, the cortical actin rim, and actomyosin-based stress fibers. This review addresses each of these actin-based structures, and discusses cellular signals that control the disposition of actin in different endothelial cell phenotypes. PMID:19028505

  6. Physical processes in an electron current layer causing intense plasma heating and formation of x-lines

    SciTech Connect

    Singh, Nagendra; Wells, B. E.; Khazanov, Igor

    2015-05-15

    We study the evolution of an electron current layer (ECL) through its several stages by means of three-dimensional particle-in-cell (PIC) simulations with ion to electron mass ratio M/m{sub e} = 400. An ECL evolves through the following stages: (i) Electrostatic (ES) current-driven instability (CDI) soon after its formation with half width w about 2 electron skin depth (d{sub e}), (ii) current disruption in the central part of the ECL by trapping of electrons and generation of anomalous resistivity, (iii) electron tearing instability (ETI) with significantly large growth rates in the lower end of the whistler frequency range, (iv) widening of the ECL and modulation of its width by the ETI, (v) gradual heating of electrons by the CDI-driven ES ion modes create the condition that the electrons become hotter than the ions, (vi) despite the reduced electron drift associated with the current disruption by the CDI, the enhanced electron temperature continues to favor a slow growth of the ion waves reaching nonlinear amplitudes, (vii) the nonlinear ion waves undergo modulation and collapse into localized density cavities containing spiky electric fields like in double layers (DLs), (viii) such spiky electric fields are very effective in further rapid heating of both electrons and ions. As predicted by the electron magnetohydrodynamic (EMHD) theories, the ETI growth rate maximizes at wave numbers in the range 0.4 < k{sub x}W < 0.8 where k{sub x} is the wave number parallel to the ECL magnetic field and w is the evolving half width of the ECL. The developing ETI generates in-plane currents that support out-of-plane magnetic fields around the emerging x-lines. The ETI and the spiky electrostatic structures are accompanied by fluctuations in the magnetic fields near and above the lower-hybrid (ion plasma) frequency, including the whistler frequency range. We compare our results with experimental results and satellite observation.

  7. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells.

    PubMed

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-09-30

    Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery.

  8. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  9. Dynamic in vivo analysis of drug induced actin cytoskeleton degradation by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Schnekenburger, Juergen; Bredebusch, Ilona; Langehanenberg, Patrik; Domschke, Wolfram; von Bally, Gert; Kemper, Björn

    2007-07-01

    The actin cytoskeleton mediates a variety of crucial cellular functions as migration, intracellular transport, exocytosis, endocytosis and force generation. The highly dynamic actin fibers are therefore targets for several drugs and toxins. However the study of actin interfering processes by standard microscopy techniques fails in the detailed resolution of dynamic spatial alterations required for a deeper understanding of toxic effects. Here we applied digital holographic microscopy in the online functional analysis of the actin cytoskeleton disrupting marine toxin Latrunculin B. SEM and fluorescence microscopy showed rapid Latrunculin B induced alterations in cell morphology and actin fiber degradation in pancreas tumor cells. The dynamic digital holographic in vivo analysis of the drug dependent cellular processes demonstrated differences in the actin cytoskeleton stability of highly differentiated and dedifferentiated pancreas tumor cell lines. The spatial resolution of the morphological alterations revealed unequal changes in cell morphology. While cells with a low metastatic potential showed Latrunculin B induced cell collapse within 4 h the metastatic tumor cells were increased in cell volume indicating Latrunculin B effects also on cell water content. These data demonstrate that marker free, non-destructive online analysis of cellular morphology and dynamic spatial processes in living cells by digital holography offers new insights in actin dependent cellular mechanisms. Digital holographic microscopy was shown to be a versatile tool in the screening of toxic drug effects and cancer cell biology.

  10. LeftyA decreases Actin Polymerization and Stiffness in Human Endometrial Cancer Cells

    PubMed Central

    Salker, Madhuri S.; Schierbaum, Nicolas; Alowayed, Nour; Singh, Yogesh; Mack, Andreas F.; Stournaras, Christos; Schäffer, Tilman E.; Lang, Florian

    2016-01-01

    LeftyA, a cytokine regulating stemness and embryonic differentiation, down-regulates cell proliferation and migration. Cell proliferation and motility require actin reorganization, which is under control of ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1). The present study explored whether LeftyA modifies actin cytoskeleton, shape and stiffness of Ishikawa cells, a well differentiated endometrial carcinoma cell line. The effect of LeftyA on globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry. Rac1 and PAK1 transcript levels were measured by qRT-PCR as well as active Rac1 and PAK1 by immunoblotting. Cell stiffness (quantified by the elastic modulus), cell surface area and cell volume were studied by atomic force microscopy (AFM). As a result, 2 hours treatment with LeftyA (25 ng/ml) significantly decreased Rac1 and PAK1 transcript levels and activity, depolymerized actin, and decreased cell stiffness, surface area and volume. The effect of LeftyA on actin polymerization was mimicked by pharmacological inhibition of Rac1 and PAK1. In the presence of the Rac1 or PAK1 inhibitor LeftyA did not lead to significant further actin depolymerization. In conclusion, LeftyA leads to disruption of Rac1 and Pak1 activity with subsequent actin depolymerization, cell softening and cell shrinkage. PMID:27404958

  11. Stochastic model of profilin-actin polymerization

    NASA Astrophysics Data System (ADS)

    Horan, Brandon; Vavylonis, Dimitrios

    A driving factor in cell motility and other processes that involve changes of cell shape is the rapid polymerization of actin subunits into long filaments. This process is regulated by profilin, a protein which binds to actin subunits and regulates elongation of actin filaments. Whether profilin stimulates polymerization by coupling to hydrolysis of ATP-bound actin is debated. Previous studies have proposed indirect coupling to ATP hydrolysis using rate equations, but did not include the effects of fluctuations that are important near the critical concentration. We developed stochastic simulations using the Gillespie algorithm to study single filament elongation at the barbed end in the presence of profilin. We used recently measured rate constants and estimated the rate of profilin binding to the barbed end such that detailed balance is satisfied. Fast phosphate release at the tip of the filament was accounted for. The elongation rate and length diffusivity as functions of profilin and actin concentration were calculated and used to extract the critical concentrations of free actin and of total actin. We show under what conditions profilin leads to an increase in the critical concentration of total actin but a decrease in the critical concentration of free actin.

  12. Contribution of nuclear actin to transcription regulation.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; Harata, Masahiko

    2015-06-01

    Actin, an integral component of the cytoskeleton, plays crucial roles in a variety of cell functions, including cell migration, adhesion, polarity and shape change. Studies performed during the last couple of decades have revealed that the actin also exists in the nucleus. However, the function and properties of nuclear actin remained elusive so far. Recently, we showed that an actin tagged with EYFP and fused with a nuclear localization signal (EYFP-NLS-actin) formed visible filamentous (F)-actin bundles in cells. To obtain further details about the individual genes that are affected by the nuclear actin, we have used the microarray analysis to determine the changes in the expression levels of RNAs in HeLa cells as a result of EYFP-NLS-actin expression. Our results suggest that the nuclear actin plays a role in the activation of genes rather than their repression. The data has been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE59799.

  13. Phosphorylation and actin activation of brain myosin.

    PubMed Central

    Barylko, B; Sobieszek, A

    1983-01-01

    A method is described for obtaining brain myosin that shows significant actin activation, after phosphorylation with chicken gizzard myosin light chain kinase. Myosin with this activity could be obtained only via the initial purification of brain actomyosin. The latter complex, isolated by a method similar to that used for smooth muscle, contained actin, myosin, tropomyosin of the non-muscle type and another actin-binding protein of approximately 100,000 daltons. From the presence of a specific myosin light chain kinase and phosphatase in brain tissue it is suggested that the regulation of actin-myosin interaction operates via phosphorylation and dephosphorylation of myosin. Images Fig. 1. Fig. 3. PMID:11894951

  14. Architecture and Connectivity Govern Actin Network Contractility.

    PubMed

    Ennomani, Hajer; Letort, Gaëlle; Guérin, Christophe; Martiel, Jean-Louis; Cao, Wenxiang; Nédélec, François; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2016-03-01

    Actomyosin contractility plays a central role in a wide range of cellular processes, including the establishment of cell polarity, cell migration, tissue integrity, and morphogenesis during development. The contractile response is variable and depends on actomyosin network architecture and biochemical composition. To determine how this coupling regulates actomyosin-driven contraction, we used a micropatterning method that enables the spatial control of actin assembly. We generated a variety of actin templates and measured how defined actin structures respond to myosin-induced forces. We found that the same actin filament crosslinkers either enhance or inhibit the contractility of a network, depending on the organization of actin within the network. Numerical simulations unified the roles of actin filament branching and crosslinking during actomyosin contraction. Specifically, we introduce the concept of "network connectivity" and show that the contractions of distinct actin architectures are described by the same master curve when considering their degree of connectivity. This makes it possible to predict the dynamic response of defined actin structures to transient changes in connectivity. We propose that, depending on the connectivity and the architecture, network contraction is dominated by either sarcomeric-like or buckling mechanisms. More generally, this study reveals how actin network contractility depends on its architecture under a defined set of biochemical conditions.

  15. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks. PMID:26915738

  16. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks.

  17. Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement.

    PubMed

    Ichikawa, Satoshi; Yamada, Noboru; Suetsugu, Noriyuki; Wada, Masamitsu; Kadota, Akeo

    2011-08-01

    The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.

  18. Actin as Deathly Switch? How Auxin Can Suppress Cell-Death Related Defence

    PubMed Central

    Chang, Xiaoli; Riemann, Michael; Liu, Qiong; Nick, Peter

    2015-01-01

    Plant innate immunity is composed of two layers – a basal immunity, and a specific effector-triggered immunity, which is often accompanied by hypersensitive cell death. Initiation of cell death depends on a complex network of signalling pathways. The phytohormone auxin as central regulator of plant growth and development represents an important component for the modulation of plant defence. In our previous work, we showed that cell death is heralded by detachment of actin from the membrane. Both, actin response and cell death, are triggered by the bacterial elicitor harpin in grapevine cells. In this study we investigated, whether harpin-triggered actin bundling is necessary for harpin-triggered cell death. Since actin organisation is dependent upon auxin, we used different auxins to suppress actin bundling. Extracellular alkalinisation and transcription of defence genes as the basal immunity were examined as well as cell death. Furthermore, organisation of actin was observed in response to pharmacological manipulation of reactive oxygen species and phospholipase D. We find that induction of defence genes is independent of auxin. However, auxin can suppress harpin-induced cell death and also counteract actin bundling. We integrate our findings into a model, where harpin interferes with an auxin dependent pathway that sustains dynamic cortical actin through the activity of phospholipase D. The antagonism between growth and defence is explained by mutual competition for signal molecules such as superoxide and phosphatidic acid. Perturbations of the auxin-actin pathway might be used to detect disturbed integrity of the plasma membrane and channel defence signalling towards programmed cell death. PMID:25933033

  19. Analysis of actinic flux profiles measured from an ozone sonde balloon

    NASA Astrophysics Data System (ADS)

    Wang, P.; Allaart, M.; Knap, W. H.; Stammes, P.

    2014-12-01

    A green light sensor has been developed at KNMI to measure actinic flux profiles using an ozone sonde balloon. In total, 63 launches with ascending and descending profiles were performed between 2006 and 2010. The measured uncalibrated actinic flux profiles are analyzed using the Doubling Adding KNMI (DAK) radiative transfer model. Values of the cloud optical thickness (COT) along the flight track were taken from the Spinning Enhanced Visible and Infrared Imager (SEVIRI) Cloud Physical Properties (CPP) product. The impact of clouds on the actinic flux profile is evaluated on the basis of the cloud modification factor (CMF) at the cloud top and cloud base, which is the ratio between the actinic fluxes for cloudy and clear-sky scenes. The impact of clouds on the actinic flux is clearly detected: the largest enhancement occurs at the cloud top due to multiple scattering. The actinic flux decreases almost linearly from cloud top to cloud base. Above the cloud top the actinic flux also increases compared to clear-sky scenes. We find that clouds can increase the actinic flux to 2.3 times of the clear-sky value at cloud top and decrease it to about 0.05 at cloud base. The relationship between CMF and COT agrees well with DAK simulations, except for a few outliers. Good agreement is found between the DAK simulated actinic flux profiles and the observations for single layer clouds in fully overcast scenes. The instrument is suitable for operational balloon measurements because of its simplicity and low cost. It is worth to further develop the instrument and launch it together with atmospheric chemistry composition sensors.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-09-15

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

  2. [Congenital myopathies - skeletal muscle diseases related to disorder of actin filament structure and functions].

    PubMed

    Robaszkiewicz, Katarzyna; Moraczewska, Joanna

    2011-01-01

    Congenital myopathies are clinically and genetically heterogeneous disorders characterized by muscle structural abnormalities, muscle weakness and deformities. The clinical spectrum of the disease ranges from severe cases with early death to adult-onset cases with slow progression. In the skeletal muscle fibers, the specific structural changes are rod-shaped structures present in the sarcoplasm (nemaline myopathy – NM) or nuclei (intranuclear rod myopathy – IRM), cap-like structures peripherally located within muscle fibers (cap disease – CD), accumulations of actin filaments (actin myopathy – AM), changes in the fiber type proportion and size (congenital fiber type disproportion – CFTD), irregularity of Z-lines and abnormal localization of myofiber nuclei. Mutations in several genes encoding muscle proteins have been linked to congenital myopathy. These genes include a-skeletal actin (ACTA1), tropomyosin (TPM2 and TPM3), troponin (TNNT1) and nebulin (NEB). In vitro and in vivo studies show that mutations identified within these genes have varying impacts on thin filament protein structure, which affect polymerization and stabilization of actin filament, actin cellular localization and regulation of actin-myosin activity. Many lines of evidence suggest that mutated proteins have "toxic" effects. Unfortunately, there is no existing simple correlation between the degree of protein disruption, muscle pathologies and disease severity. PMID:21677359

  3. Force of an actin spring

    NASA Astrophysics Data System (ADS)

    Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul

    2003-03-01

    The acrosomal process of the horseshoe crab sperm is a novel mechanochemical molecular spring that converts its elastic stain energy to mechanical work upon the chemical activation by Ca2+. Twisted and bent, the initial state of the acrosomal bundle features a high degree of complexity in its structure and the energy is believed to be stored in the highly strained actin filaments as an elastic potential energy. When activated, the bundle relaxes from the coil of the highly twisted and bent filaments to its straight conformation at a mean velocity of 15um/s. The mean extension velocity increases dramatically from 3um/s to 27um/s when temperature of the medium is changed from 9.6C to 32C (respective viscosities of 1.25-0.75cp), yet it exhibits a very weak dependence on changes in the medium viscosity (1cp-33cp). These experiments suggest that the uncoiling of the actin spring should be limited not by the viscosity of the medium but by the unlatching events of involved proteins at a molecular level. Unlike the viscosity-limited processes, where force is directly related to the rate of the reaction, a direct measurement is required to obtain the spring force of the acrosomal process. The extending acrosomal bundle is forced to push against a barrier and its elastic buckling response is analyzed to measure the force generated during the uncoiling.

  4. Structural, Mechanical, and Dynamical Variability of the Actin Cortex in Living Cells

    PubMed Central

    Eghiaian, Frédéric; Rigato, Annafrancesca; Scheuring, Simon

    2015-01-01

    In eukaryotic cells, an actin-based cortex lines the inner leaflet of the plasma membrane, endowing the cells with crucial mechanical and functional properties. Unfortunately, it has not been possible to study the structural dynamics of the actin cortex at high lateral resolution in living cells. Here, we performed atomic force microscopy time-lapse imaging and mechanical mapping of actin in the cortex of living cells at high lateral and temporal resolution. Cortical actin filaments adopted discernible arrangements, ranging from large parallel bundles with low connectivity to a tight meshwork of short filaments. Mixing of these architectures resulted in attuned cortex networks with specific connectivity, mechanical responses, and marked differences in their dynamic behavior. PMID:25809247

  5. A model for transition by attachment-line contamination and an examination of cross-flow instability in 3-D boundary layers

    NASA Astrophysics Data System (ADS)

    Stewart, I. R.; Poll, D. I. A.

    A model for predicting the development of intermittent turbulence in a boundary layer flow downstream of a large 2-D circular trip on an attachment-line was produced. It gives good predictions of intermittency at any point in the flow. Stability calculations were performed for the Falkner-Skan-Cooke family of boundary layers using linear stability theory. A relationship for the minimum critical Reynolds number and the height of the inflection point in the profile is identified. This correlation gives good agreement with the stability characteristics for boundary layer profiles typical of those occurring on suction wings. A discrepancy in previous empirical relations between skin friction and Reynolds number in fully developed turbulent flow on an attachment-line is discovered. To investigate this an experiment was undertaken using Preston tubes and a Pitot tube with the Clauser Chart method. An accurate correlation of skin friction on an attachment-line was determined from the experimental data. Small diameter Preston tubes underestimate skin friction on an attachment-line to the order of 10 percent which is not inconsistent with a reduction of the effective measurement height, possibly due to vortex shedding in the divergent flow at the mouth of the tube.

  6. THE TRANSITION LAYER BETWEEN THE THIN ACCRETION DISK AND THE HOT CORONA: CONSTRAINTS FROM THE THERMAL X-RAY LINE EMISSION

    SciTech Connect

    Xu, Ya-Di

    2013-02-15

    The observed continuum spectra in the low-luminosity active galactic nuclei, M81, can be reproduced by the advection-dominated accretion flow (ADAF)+thin disk/corona accretion flow model. We find that the best fit to the spectral energy distribution of M81 requires accretion rate m-dot =3 Multiplication-Sign 10{sup -4} for R {sub tr} = 60 R {sub S}. The observed H-like and He-like iron lines may probably be emitted from the transition layer between the vertically connected hot corona and cold thin disk. We model a power-law function of height z for the electron temperature in the transition layer, and calculate the thermal H-like and He-like iron line emission from M81. Combining the observations with our results, we find that the thickness of the transition layer is {approx}14%-23% of the thickness of the corona at the same radius. We discuss that the observed stronger H-like line emission than He-like line emission cannot be explained with the pure ADAF model. Our results provide useful constraints on the theoretical model for disk-corona connection.

  7. Effect of alpha-actinin on actin structure. Actin ATPase activity.

    PubMed

    Singh, I; Goll, D E; Robson, R M

    1981-08-28

    Alpha-Actinin increases the ATPase activity of actin by up to 84%, depending un pH, divalent cations present and the added Mg2+: ATP ratio. Dithiothreitol decreases actin ATPase activity approx. 20% but does not reduce the ability of alpha-actinin to increase actin ATP activity. Increasing amounts of added alpha-actinin up to 1 mos alpha-actinin to 49 mol actin cause in increasing increment in actin ATPase activity, but adding alpha-actinin beyond 1 mol alpha-actinin to 49 mol actin elicits only small additional increments in activity. Actin ATPase activity ranges from approx 100 nmol Pi/mg actin per h (4.3 mol Pi/mol actin per h) at high levels (10 mM) of ATP in the presence of lower amounts (1 mM) of added mg2+ to approx. 12.5 nmol Pi/mg actin per h (0.52 mol Pi/mol actin per h) at high pH (8.5) or at low levels (0.5-1.0 mM) of ATP in the presence of higher amounts (10 mM) of added Mg2+ ATp uncomplexed with Mg2+ inhibits the ability of alpha-actinin to increase F-actin ATPase activity. Activities with different divalent cations showed that the actin ATPase in these studies, which was 1/100 as great as Mg2+-modified actomyosin ATPase activity, was not due to trace amounts of myosin contaminating the actin preparations. The results are consistent with the concept that alpha-actinin can alter the structure of actin monomers. PMID:6456018

  8. Change in the actin-myosin subfragment 1 interaction during actin polymerization.

    PubMed

    Chaussepied, P; Kasprzak, A A

    1989-12-01

    To better characterize the conformational differences of G- and F-actin, we have compared the interaction between G- and F-actin with myosin subfragment 1 (S1) which had part of its F-actin binding site (residues 633-642) blocked by a complementary peptide or "antipeptide" (Chaussepied, P., and Morales, M. F. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471-7475). Light scattering, sedimentation, and electron microscopy measurements showed that, with the antipeptide covalently attached to the S1 heavy chain, S1 was not capable of inducing G-actin polymerization in the absence of salt. Moreover, the antipeptide-carrying S1 did not change the fluorescence polarization of 5-[2-(iodoacetyl)-aminoethyl]aminonaphthalene-1-sulfonic acid (1,5-IAEDANS)-labeled G-actin or of 1,5-IAEDANS-labeled actin dimer, compared to the control S1. This result, interpreted as a lack of interaction between G-actin and antipeptide-carrying S1, was confirmed further by the following experiments: in the presence of G-actin, antipeptide.S1 heavy chain was not protected against trypsin and papain proteolysis, and G-actin could not be cross-linked to antipeptide.S1 by 1-ethyl-3[-3-(dimethylamino)propyl]carbodiimide. In contrast, similar experiments showed that antipeptide.S1 was able to interact with nascent F-actin and with F-actin. Thus, blocking the stretch 633-642 of S1 heavy chain by the antipeptide strongly inhibits G-actin-S1 interaction but only slightly alters F-actin-S1 contact. We, therefore postulate that this stretch of skeletal S1 heavy chain is essential for G-actin-S1 interaction and that the G-F transformation generates new S1 binding site(s) on the actin molecule.

  9. Scrambling of the actin I gene in two Oxytricha species.

    PubMed Central

    DuBois, M; Prescott, D M

    1995-01-01

    The DNA in a germ-line nucleus (a micronucleus) undergoes extensive processing when it develops into a somatic nucleus (a macronucleus) after cell mating in hypotrichous ciliates. Processing includes destruction of a large amount of spacer DNA between genes and excision of gene-sized molecules from chromosomes. Before processing, micronuclear genes are interrupted by numerous noncoding segments called internal eliminated sequences (IESs). The IESs are excised and destroyed, and the retained macro-nuclear-destined sequences (MDSs) are spliced. MDSs in some micronuclear genes are not in proper order and must be reordered during processing to create functional gene-sized molecules for the macronucleus. Here we report that the micronuclear actin I gene in Oxytricha trifallax WR consists of 10 MDSs and 9 IESs compared to the previously reported 9 MDSs and 8 IESs in the micronuclear actin I gene of Oxytricha nova. The MDSs in the actin I gene are scrambled in a similar pattern in the two species, but the positions of MDS-IES junctions are shifted by up to 14 bp for scrambled and 138 bp for the nonscrambled MDSs. The shifts in MDS-IES junctions create differences in the repeat sequences that are believed to guide MDS splicing. Also, the sizes and sequences of IESs in the micronuclear actin I genes are different in the two Oxytricha species. These observations give insight about the possible origins of IES insertion and MDS scrambling in evolution and show the extraordinary malleability of the germ-line DNA in hypotrichs. Images Fig. 2 Fig. 3 Fig. 4 PMID:7732002

  10. Unconventional actin conformations localize on intermediate filaments in mitosis

    SciTech Connect

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-03-04

    Research highlights: {yields} Unconventional actin conformations colocalize with vimentin on a cage-like structure in metaphase HEK 293T cells. {yields} These conformations are detected with the anti-actin antibodies 1C7 ('lower dimer') and 2G2 ('nuclear actin'), but not C4 (monomeric actin). {yields} Mitotic unconventional actin cables are independent of filamentous actin or microtubules. {yields} Unconventional actin colocalizes with vimentin on a nocodazole-induced perinuclear dense mass of cables. -- Abstract: Different structural conformations of actin have been identified in cells and shown to reside in distinct subcellular locations of cells. In this report, we describe the localization of actin on a cage-like structure in metaphase HEK 293T cells. Actin was detected with the anti-actin antibodies 1C7 and 2G2, but not with the anti-actin antibody C4. Actin contained in this structure is independent of microtubules and actin filaments, and colocalizes with vimentin. Taking advantage of intermediate filament collapse into a perinuclear dense mass of cables when microtubules are depolymerized, we were able to relocalize actin to such structures. We hypothesize that phosphorylation of intermediate filaments at mitosis entry triggers the recruitment of different actin conformations to mitotic intermediate filaments. Storage and partition of the nuclear actin and antiparallel 'lower dimer' actin conformations between daughter cells possibly contribute to gene transcription and transient actin filament dynamics at G1 entry.

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

    DOEpatents

    Sederoff, Heike; Huber, Steven C; Larabell, Carolyn A

    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.

  12. Profilin connects actin assembly with microtubule dynamics.

    PubMed

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

    2016-08-01

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

  13. Probing the actin-auxin oscillator

    PubMed Central

    2010-01-01

    The directional transport of the plant hormone auxin depends on transcellular gradients of auxin-efflux carriers that continuously cycle between plasma membrane and intracellular compartments. This cycling has been proposed to depend on actin filaments. However, the role of actin for the polarity of auxin transport has been disputed. To get insight into this question, actin bundling was induced by overexpression of the actin-binding domain of talin in tobacco BY-2 cells and in rice plants. This bundling can be reverted by addition of auxins, which allows to address the role of actin organization on the flux of auxin. In both systems, the reversion of a normal actin configuration can be restored by addition of exogenous auxins and this fully restores the respective auxin-dependent functions. These findings lead to a model of a self-referring regulatory circuit between polar auxin transport and actin organization. To further dissect the actin-auxin oscillator, we used photoactivated release of caged auxin in tobacco cells to demonstrate that auxin gradients can be manipulated at a subcellular level. PMID:20023411

  14. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

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

    2016-01-01

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

  15. Actin cytoskeleton redox proteome oxidation by cadmium

    PubMed Central

    Go, Young-Mi; Orr, Michael

    2013-01-01

    Epidemiological studies associate environmental cadmium (Cd) exposure with the risk of lung diseases. Although mechanisms are not fully elucidated, several studies demonstrate Cd effects on actin and actin-associated proteins. In a recent study of Cd at concentrations similar to environmental exposures, we found that redox-dependent inflammatory signaling by NF-κB was sensitive to the actin-disrupting agent, cytochalasin D. The goal of the present study was to use mass spectrometry-based redox proteomics to investigate Cd effects on the actin cytoskeleton proteome and related functional pathways in lung cells at low environmental concentrations. The results showed that Cd under conditions that did not alter total protein thiols or glutathione redox state caused significant oxidation of peptidyl Cys of proteins regulating actin cytoskeleton. Immunofluorescence microscopy of lung fibroblasts and pulmonary artery endothelial cells showed that low-dose Cd exposure stimulated filamentous actin formation and nuclear localization of destrin, an actin-depolymerizing factor. Taken together, the results show that redox states of peptidyl Cys in proteins associated with actin cytoskeleton pathways are selectively oxidized in lung by Cd at levels thought to occur from environmental exposure. PMID:24077948

  16. Colchicine activates actin polymerization by microtubule depolymerization.

    PubMed

    Jung, H I; Shin, I; Park, Y M; Kang, K W; Ha, K S

    1997-06-30

    Swiss 3T3 fibroblasts were treated with the microtubule-disrupting agent colchicine to study any interaction between microtubule dynamics and actin polymerization. Colchicine increased the amount of filamentous actin (F-actin), in a dose- and time-dependent manner with a significant increase at 1 h by about 130% over control level. Confocal microscopic observation showed that colchicine increased F-actin contents by stress fiber formation without inducing membrane ruffling. Colchicine did not activate phospholipase C and phospholipase D, whereas lysophosphatidic acid did, indicating that colchicine may have a different mechanism of actin polymerization regulation from LPA. A variety of microtubule-disrupting agents stimulated actin polymerization in Swiss 3T3 and Rat-2 fibroblasts as did colchicine, but the microtubule-stabilizing agent taxol inhibited actin polymerization induced by the above microtubule-disrupting agents. In addition, colchicine-induced actin polymerization was blocked by two protein phosphatase inhibitors, okadaic acid and calyculin A. These results suggest that microtubule depolymerization activates stress fiber formation by serine/threonine dephosphorylation in fibroblasts. PMID:9264034

  17. Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network

    PubMed Central

    Luo, Weiwei; Lieu, Zi Zhao; Manser, Ed; Bershadsky, Alexander D.; Sheetz, Michael P.

    2016-01-01

    A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization. PMID:27760153

  18. Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks.

    PubMed

    Lappalainen, Pekka

    2016-08-15

    The actin cytoskeleton supports a vast number of cellular processes in nonmuscle cells. It is well established that the organization and dynamics of the actin cytoskeleton are controlled by a large array of actin-binding proteins. However, it was only 40 years ago that the first nonmuscle actin-binding protein, filamin, was identified and characterized. Filamin was shown to bind and cross-link actin filaments into higher-order structures and contribute to phagocytosis in macrophages. Subsequently many other nonmuscle actin-binding proteins were identified and characterized. These proteins regulate almost all steps of the actin filament assembly and disassembly cycles, as well as the arrangement of actin filaments into diverse three-dimensional structures. Although the individual biochemical activities of most actin-regulatory proteins are relatively well understood, knowledge of how these proteins function together in a common cytoplasm to control actin dynamics and architecture is only beginning to emerge. Furthermore, understanding how signaling pathways and mechanical cues control the activities of various actin-binding proteins in different cellular, developmental, and pathological processes will keep researchers busy for decades. PMID:27528696

  19. Integration of linear and dendritic actin nucleation in Nck-induced actin comets

    PubMed Central

    Borinskaya, Sofya; Velle, Katrina B.; Campellone, Kenneth G.; Talman, Arthur; Alvarez, Diego; Agaisse, Hervé; Wu, Yi I.; Loew, Leslie M.; Mayer, Bruce J.

    2016-01-01

    The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails—dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens. PMID:26609071

  20. Integration of linear and dendritic actin nucleation in Nck-induced actin comets.

    PubMed

    Borinskaya, Sofya; Velle, Katrina B; Campellone, Kenneth G; Talman, Arthur; Alvarez, Diego; Agaisse, Hervé; Wu, Yi I; Loew, Leslie M; Mayer, Bruce J

    2016-01-15

    The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails--dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens. PMID:26609071

  1. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  2. Crystal structure of an archaeal actin homolog.

    PubMed

    Roeben, Annette; Kofler, Christine; Nagy, István; Nickell, Stephan; Hartl, F Ulrich; Bracher, Andreas

    2006-04-21

    Prokaryotic homologs of the eukaryotic structural protein actin, such as MreB and ParM, have been implicated in determination of bacterial cell shape, and in the segregation of genomic and plasmid DNA. In contrast to these bacterial actin homologs, little is known about the archaeal counterparts. As a first step, we expressed a predicted actin homolog of the thermophilic archaeon Thermoplasma acidophilum, Ta0583, and determined its crystal structure at 2.1A resolution. Ta0583 is expressed as a soluble protein in T.acidophilum and is an active ATPase at physiological temperature. In vitro, Ta0583 forms sheets with spacings resembling the crystal lattice, indicating an inherent propensity to form filamentous structures. The fold of Ta0583 contains the core structure of actin and clearly belongs to the actin/Hsp70 superfamily of ATPases. Ta0583 is approximately equidistant from actin and MreB on the structural level, and combines features from both eubacterial actin homologs, MreB and ParM. The structure of Ta0583 co-crystallized with ADP indicates that the nucleotide binds at the interface between the subdomains of Ta0583 in a manner similar to that of actin. However, the conformation of the nucleotide observed in complex with Ta0583 clearly differs from that in complex with actin, but closely resembles the conformation of ParM-bound nucleotide. On the basis of sequence and structural homology, we suggest that Ta0583 derives from a ParM-like actin homolog that was once encoded by a plasmid and was transferred into a common ancestor of Thermoplasma and Ferroplasma. Intriguingly, both genera are characterized by the lack of a cell wall, and therefore Ta0583 could have a function in cellular organization.

  3. Computational defect review for actinic mask inspections

    NASA Astrophysics Data System (ADS)

    Morgan, Paul; Rost, Daniel; Price, Daniel; Corcoran, Noel; Satake, Masaki; Hu, Peter; Peng, Danping; Yonenaga, Dean; Tolani, Vikram

    2013-04-01

    As optical lithography continues to extend into low-k1 regime, resolution of mask patterns continues to diminish. The limitation of 1.35 NA posed by water-based lithography has led to the application of various resolution enhancement techniques (RET), for example, use of strong phase-shifting masks, aggressive OPC and sub-resolution assist features, customized illuminators, etc. The adoption of these RET techniques combined with the requirements to detect even smaller defects on masks due to increasing MEEF, poses considerable challenges for a mask inspection engineer. Inspecting masks under their actinic-aerial image conditions would detect defects that are more likely to print under those exposure conditions. However, this also makes reviewing such defects in their low-contrast aerial images very challenging. On the other hand, inspecting masks under higher resolution inspection optics would allow for better viewing of defects post-inspection. However, such inspections generally would also detect many more defects, including printable and nuisance, thereby making it difficult to judge which are of real concern for printability on wafer. Often, an inspection engineer may choose to use Aerial and/or high resolution inspection modes depending on where in the process flow the mask is and the specific device-layer characteristics of the mask. Hence, a comprehensive approach is needed in handling defects both post-aerial and post-high resolution inspections. This analysis system is designed for the Applied Materials Aera™ mask inspection platform, all data reported was collected using the Aera.

  4. Asymmetry of the Balmer-alpha line shape and recovery of the effective hydrogen temperature in the tokamak scrape-off layer

    SciTech Connect

    Neverov, V. S. Kukushkin, A. B.; Lisgo, S. W. Kukushkin, A. S.; Alekseev, A. G.

    2015-02-15

    An algorithm for recovering the effective temperature of atoms of hydrogen (and its isotopes) in the tokamak scrape-off layer from the asymmetry of the Balmer-alpha line shape is proposed. The algorithm is based on the parametrization of the asymmetry of the line shape caused by the nonlocal character of neutral hydrogen flux from the wall into the tokamak plasma. The accuracy of the algorithm is tested against the results of simulations of the velocity distribution function of deuterium neutrals in the scrape-off layer by the EIRENE code with the use of the source data on the main plasma component in the quasi-stationary stage of the inductive mode of ITER operation calculated by the SOLPS4.3 (B2-EIRENE) code.

  5. Probing actin incorporation into myofibrils using Asp11 and His73 actin mutants.

    PubMed

    Xia, D; Peng, B; Sesok, D A; Peng, I

    1993-01-01

    We used a cell free system Bouché et al.: J. Cell Biol. 107:587-596, 1988] to study the incorporation of actin into myofibrils. We used alpha-skeletal muscle actin and actins with substitutions of either His73 [Solomon and Rubenstein: J. Biol.Chem. 262:11382, 1987], or Asp11 [Solomon et al.: J. Biol. Chem. 263:19662, 1988]. Actins were translated in reticulocyte lysate and incubated with myofibrils. The incorporated wild type actin could be cross-linked into dimers using N,N'-1,4-phenylenebismaleimide (PBM), indicating that the incorporated actin is actually inserted into the thin filaments of the myofibril. The His73 mutants incorporated to the same extent as wild type actin and was also cross-linked with PBM. Although some of the Asp11 mutants co-assembled with carrier actin, only 1-3% of the Asp11 mutant actins incorporated after 2 min and did not increase after 2 hr. Roughly 17% of wild type actin incorporated after 2 min and 31% after 2 hr. ATP increased the release of wild type actin from myofibrils, but did not increase the release of Asp11 mutants. We suggest that (1) the incorporation of wild type and His73 mutant actins was due to a physiological process whereas association of Asp11 mutants with myofibrils was non-specific, (2) the incorporation of wild type actin involved a rapid initial phase, followed by a slower phase, and (3) since some of the Asp11 mutants can co-assemble with wild type actin, the ability to self-assemble was not sufficient for incorporation into myofibrils. Thus, incorporation probably includes interaction between actin and a thin filament associated protein. We also showed that incorporation occurred at actin concentrations which would cause disassembly of F-actin. Since the myofibrils did not show large scale disassembly but incorporated actin, filament stability and monomer incorporation are likely to be mediated by actin associated proteins of the myofibril. PMID:8287497

  6. Comparison of semen variables, sperm DNA damage and sperm membrane proteins in two male layer breeder lines.

    PubMed

    M, Shanmugam; T R, Kannaki; A, Vinoth

    2016-09-01

    Semen variables are affected by the breed and strain of chicken. The present study was undertaken to compare the semen quality in two lines of adult chickens with particular reference to sperm chromatin condensation, sperm DNA damage and sperm membrane proteins. Semen from a PD3 and White Leghorn control line was collected at 46 and 47 weeks and 55 weeks of age. The semen was evaluated for gross variables and sperm chromatin condensation by aniline blue staining. Sperm DNA damage was assessed by using the comet assay at 47 weeks of age and sperm membrane proteins were assessed at 55 weeks of age. The duration of fertility was studied by inseminating 100 million sperm once into the hens of the same line as well as another line. The eggs were collected after insemination for 15days and incubated. The eggs were candled on 18th day of incubation for observing embryonic development. The White Leghorn control line had a greater sperm concentration and lesser percentage of morphologically abnormal sperm at the different ages where assessments occurred. There was no difference in sperm chromatin condensation, DNA damage and membrane proteins between the lines. Only low molecular weight protein bands of less than 95kDa were observed in samples of both lines. The line from which semen was used had no effect on the duration over which fertility was sustained after insemination either when used in the same line or another line. Thus, from the results of the present study it may be concluded that there was a difference in gross semen variables between the lines that were studied, however, the sperm chromatin condensation, DNA damage, membrane proteins and duration over which fertility was sustained after insemination did not differ between the lines. PMID:27470200

  7. Comparison of semen variables, sperm DNA damage and sperm membrane proteins in two male layer breeder lines.

    PubMed

    M, Shanmugam; T R, Kannaki; A, Vinoth

    2016-09-01

    Semen variables are affected by the breed and strain of chicken. The present study was undertaken to compare the semen quality in two lines of adult chickens with particular reference to sperm chromatin condensation, sperm DNA damage and sperm membrane proteins. Semen from a PD3 and White Leghorn control line was collected at 46 and 47 weeks and 55 weeks of age. The semen was evaluated for gross variables and sperm chromatin condensation by aniline blue staining. Sperm DNA damage was assessed by using the comet assay at 47 weeks of age and sperm membrane proteins were assessed at 55 weeks of age. The duration of fertility was studied by inseminating 100 million sperm once into the hens of the same line as well as another line. The eggs were collected after insemination for 15days and incubated. The eggs were candled on 18th day of incubation for observing embryonic development. The White Leghorn control line had a greater sperm concentration and lesser percentage of morphologically abnormal sperm at the different ages where assessments occurred. There was no difference in sperm chromatin condensation, DNA damage and membrane proteins between the lines. Only low molecular weight protein bands of less than 95kDa were observed in samples of both lines. The line from which semen was used had no effect on the duration over which fertility was sustained after insemination either when used in the same line or another line. Thus, from the results of the present study it may be concluded that there was a difference in gross semen variables between the lines that were studied, however, the sperm chromatin condensation, DNA damage, membrane proteins and duration over which fertility was sustained after insemination did not differ between the lines.

  8. Up-regulation of drug-metabolizing enzyme genes in layered co-culture of a human liver cell line and endothelial cells.

    PubMed

    Ohno, Maki; Motojima, Kiyoto; Okano, Teruo; Taniguchi, Akiyoshi

    2008-11-01

    Primary human hepatocytes are used extensively to study drug-metabolizing enzymes such as the cytochrome P450 (CYP) enzymes. However, the activities of these enzymes decrease rapidly during culture. In the present study, using a thermo-responsive culture dish, layered co-culture was achieved by placing a bovine pulmonary artery endothelial cell (BPAEC) sheet onto the human hepatoma cell line HepG2. In the BPAEC/HepG2 layered co-culture system, real-time polymerase chain reaction analysis showed that the expression levels of various CYP enzymes were more than 10 times greater 21 days after layering than with a HepG2 monolayer. The expression levels of CYP1B1, CYP2C9, CYP2E1, and CYP3A4 were up-regulated in a time-dependent manner, gradually increasing from day 10 after layering, and continuing to increase until at least day 21. The gene expression levels of the various CYP enzymes were almost identical to that of human liver. These results suggest that our layered co-culture system enhances the function of HepG2 cells and that our BPAEC/HepG2 layered co-culture system can serve as a useful model for the in vitro evaluation of CYP regulation. PMID:18847370

  9. Dynamics of an actin spring

    NASA Astrophysics Data System (ADS)

    Riera, Christophe; Mahadevan, L.; Shin, Jennifer; Matsudaira, Paul

    2003-03-01

    The acrosome of the sperm of the horseshoe crab (Limulus Polyphemus) is an unusual actin based system that shows a spectacular dynamical transition in the presence of Ca++ that is present in abundance in the neighborhood of the egg. During this process, the bundle, which is initially bent and twisted uncoils and becomes straight in a matter of a few seconds. Based on microstructural data, we propose a model for the dynamics of uncoiling that is best represented by a triple-well potential corresponding to the different structural arrangements of the supertwisted filaments. Each of the false, true and coiled states corresponds to a local minimum of the energy, with the true state being the one with the lowest energy. Using an evolution equation derived by balancing torques, we investigate the nucleation and propagation of the phase transition and compare the results with those of experiments. Our model quantifies the hypothesis that the acrosomal bundle behaves like a mechano-chemical spring.

  10. Middle-term expansion of hematopoietic cord blood cells with new human stromal cell line feeder-layers and different cytokine cocktails.

    PubMed

    De Angeli, S; Baiguera, S; Del Pup, L; Pavan, E; Gajo, G B; Di Liddo, R; Conconi, M T; Grandi, C; Schiavon, O; Parnigotto, P P

    2009-12-01

    Cord blood (CB) is a source of hematopoietic stem cells (HSCs) and is an alternative to bone marrow for allogenic transplantation in patients with hematological disorders. The improvement of HSC in vitro expansion is one of the main challenges in cell therapy. Stromal components and soluble factors, such as cytokines, can be useful to induce in vitro cell expansion. Hence, we investigated whether feeder-layers from new stromal cell lines and different exogenous cytokine cocktails induce HSC expansion in middle-term cultures. CB HSC middle-term expansion was carried out in co-cultures on different feeder-layers exposed to three different cytokine cocktails. CB HSC expansion was also carried out in stroma-free cultures in the presence of different cytokine cocktails. Clonogenic tests were performed, and cell growth levels were evaluated. Moreover, the presence of VCAM-1 mRNA was assessed, and the mesenchymal cell-like phenotype expression was detected. All feeder-layers were able to induce a significant clonogenic growth with respect to the control culture, and all of the cytokine cocktails induced a significant increase in CB cell expansion indexes, even though no potential variation dependent on their composition was noted. The modulative effects of the different cocktails, exerted on each cell line used, was dependent on their composition. Finally, all cell lines were positive for CD73, CD117 and CD309, similar to mesenchymal stem cells present in adult bone marrow and in other human tissues, and negative for the hematopoietic markers. These data indicate that our cell lines have, not only a stromal cell-like phenotype, but also a mesenchymal cell-like phenotype, and they have the potential to support in vitro expansion of CB HSCs. Moreover, exogenous cytokines can be used in synergism with feeder-layers to improve the expansion levels of CB HSCs in preparation for their clinical use in allogenic transplantation. PMID:19885627

  11. Cell-specific activation of the human skeletal alpha-actin by androgens.

    PubMed

    Hong, Mei Hua; Sun, Hong; Jin, Cheng He; Chapman, Mark; Hu, Junlian; Chang, William; Burnett, Kelven; Rosen, Jon; Negro-Vilar, Andres; Miner, Jeffrey N

    2008-03-01

    Although it is evident that androgens increase muscle mass and strength, little is known about the critical molecular targets of androgens in skeletal muscle. In rodents, the skeletal alpha-actin gene is a tissue-specific gene expressed only in the levator ani and other skeletal muscles but not in the prostate or preputial gland, the well-known androgen target tissue. We identified tissue-specific androgen-regulated genes in the skeletal muscle in rats after oral administration of androgens and focused on androgen-dependent up-regulation of the skeletal alpha-actin gene. To investigate the mechanism of action, an in vitro system with various cell lines and a series of deletion mutants of the alpha-actin promoter were used. The human skeletal alpha-actin promoter was activated by androgens in the muscle cell line C2C12 but not in the liver, prostate, or breast cancer cell lines in which exogenous human androgen receptor is expressed. The sequence of the promoter is sufficient for cell-specific androgen response, providing a model for the tissue specificity demonstrated in vivo. Using a series of deletion mutants, the androgen response can be maintained using just the proximal promoter region. The importance of androgen regulation of this small portion of the human skeletal alpha-actin promoter was demonstrated by the correlation between muscle and the alpha-actin promoter activity for an array of selective androgen receptor modulators (SARMs), including an orally active SARM LGD2226. Taken together, the results suggest that the regulation of skeletal alpha-actin by androgens/SARMs may represent an important model system for understanding androgen anabolic action in the muscle.

  12. Strong Dependence of Hydration State of F-Actin on the Bound Mg(2+)/Ca(2+) Ions.

    PubMed

    Suzuki, Makoto; Imao, Asato; Mogami, George; Chishima, Ryotaro; Watanabe, Takahiro; Yamaguchi, Takaya; Morimoto, Nobuyuki; Wazawa, Tetsuichi

    2016-07-21

    Understanding of the hydration state is an important issue in the chemomechanical energetics of versatile biological functions of polymerized actin (F-actin). In this study, hydration-state differences of F-actin by the bound divalent cations are revealed through precision microwave dielectric relaxation (DR) spectroscopy. G- and F-actin in Ca- and Mg-containing buffer solutions exhibit dual hydration components comprising restrained water with DR frequency f2 (fw). The hydration state of F-actin is strongly dependent on the ionic composition. In every buffer tested, the HMW signal Dhyme (≡ (f1 - fw)δ1/(fwδw)) of F-actin is stronger than that of G-actin, where δw is DR-amplitude of bulk solvent and δ1 is that of HMW in a fixed-volume ellipsoid containing an F-actin and surrounding water in solution. Dhyme value of F-actin in Ca2.0-buffer (containing 2 mM Ca(2+)) is markedly higher than in Mg2.0-buffer (containing 2 mM Mg(2+)). Moreover, in the presence of 2 mM Mg(2+), the hydration state of F-actin is changed by adding a small fraction of Ca(2+) (∼0.1 mM) and becomes closer to that of the Ca-bound form in Ca2.0-buffer. This is consistent with the results of the partial specific volume and the Cotton effect around 290 nm in the CD spectra, indicating a change in the tertiary structure and less apparent change in the secondary structure of actin. The number of restrained water molecules per actin (N2) is estimated to be 1600-2100 for Ca2.0- and F-buffer and ∼2500 for Mg2.0-buffer at 10-15 °C. These numbers are comparable to those estimated from the available F-actin atomic structures as in the first water layer. The number of HMW molecules is roughly explained by the volume between the equipotential surface of -kT/2e and the first water layer of the actin surface by solving the Poisson-Boltzmann equation using UCSF Chimera. PMID:27332748

  13. Effects of Repeated Social Disruption on the Serotonergic and Dopaminergic Systems in two Genetic Lines of White Leghorn Layers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study was designed to examine whether there are genetic differences in response to repeated social disruption (RSD). Two genetic strains of White Leghorn hens were used in the study; i.e., HGPS (the line selected for high group production and survivability), and DXL (DeKalb XL commercial line). ...

  14. Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

    PubMed Central

    Mehta, Simren; Sibley, L. David

    2011-01-01

    Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion. PMID:21346192

  15. Reversible stress softening of actin networks

    NASA Astrophysics Data System (ADS)

    Chaudhuri, Ovijit; Parekh, Sapun H.; Fletcher, Daniel A.

    2007-01-01

    The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.

  16. Radial profile measurement of electron temperature in edge stochastic magnetic field layer of LHD using intensity ratio of extreme ultraviolet line emissions

    SciTech Connect

    Wang Erhui; Morita, Shigeru; Kobayashi, Masahiko; Murakami, Izumi; Goto, Motoshi; Dong Chunfeng

    2012-10-15

    Vertical profile of neon line emissions in 30-650 A wavelength range has been observed in horizontally elongated plasma cross section of Large Helical Device (LHD). Intensity ratio between the neon line emissions is studied to measure the radial profile of electron temperature in the edge stochastic magnetic field layer of LHD. The edge temperature profile successfully obtained from the line ratio of NeVIII 2s-3p to 2p-3s transitions is compared with the simulation based on three-dimensional edge transport code. The result shows a reasonably good agreement with the edge temperature profile analyzed from atomic data and analysis structure code. The electron temperature at last closed flux surface measured from the intensity ratio is also in good agreement with that measured from Thomson scattering.

  17. Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra-Golgi pH.

    PubMed

    Lázaro-Diéguez, Francisco; Jiménez, Nuria; Barth, Holger; Koster, Abraham J; Renau-Piqueras, Jaime; Llopis, Juan L; Burger, Koert N J; Egea, Gustavo

    2006-12-01

    Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H+ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis.

  18. EXTRACTION AND ANALYSIS OF ACTIN NETWORKS BASED ON OPEN ACTIVE CONTOUR MODELS

    PubMed Central

    Xu, Ting; Li, Hongsheng; Shen, Tian; Ojkic, Nikola; Vavylonis, Dimitrios; Huang, Xiaolei

    2011-01-01

    Network structures formed by actin filaments are present in many kinds of fluorescence microscopy images. In order to quantify the conformations and dynamics of such actin filaments, we propose a fully automated method to extract actin networks from images and analyze network topology. The method handles well intersecting filaments and, to some extent, overlapping filaments. First we automatically initialize a large number of Stretching Open Active Contours (SOACs) from ridge points detected by searching for plus-to-minus sign changes in the gradient map of the image. These initial SOACs then elongate simultaneously along the bright center-lines of filaments by minimizing an energy function. During their evolution, they may merge or stop growing, thus forming a network that represents the topology of the filament ensemble. We further detect junction points in the network and break the SOACs at junctions to obtain “SOAC segments”. These segments are then re-grouped using a graph-cut spectral clustering method to represent the configuration of actin filaments. The proposed approach is generally applicable to extracting intersecting curvilinear structures in noisy images. We demonstrate its potential using two kinds of data: (1) actin filaments imaged by Total Internal Reflection Fluorescence Microscopy (TIRFM) in vitro; (2) actin cytoskeleton networks in fission yeast imaged by spinning disk confocal microscopy. PMID:21822463

  19. EXTRACTION AND ANALYSIS OF ACTIN NETWORKS BASED ON OPEN ACTIVE CONTOUR MODELS.

    PubMed

    Xu, Ting; Li, Hongsheng; Shen, Tian; Ojkic, Nikola; Vavylonis, Dimitrios; Huang, Xiaolei

    2011-03-30

    Network structures formed by actin filaments are present in many kinds of fluorescence microscopy images. In order to quantify the conformations and dynamics of such actin filaments, we propose a fully automated method to extract actin networks from images and analyze network topology. The method handles well intersecting filaments and, to some extent, overlapping filaments. First we automatically initialize a large number of Stretching Open Active Contours (SOACs) from ridge points detected by searching for plus-to-minus sign changes in the gradient map of the image. These initial SOACs then elongate simultaneously along the bright center-lines of filaments by minimizing an energy function. During their evolution, they may merge or stop growing, thus forming a network that represents the topology of the filament ensemble. We further detect junction points in the network and break the SOACs at junctions to obtain "SOAC segments". These segments are then re-grouped using a graph-cut spectral clustering method to represent the configuration of actin filaments. The proposed approach is generally applicable to extracting intersecting curvilinear structures in noisy images. We demonstrate its potential using two kinds of data: (1) actin filaments imaged by Total Internal Reflection Fluorescence Microscopy (TIRFM) in vitro; (2) actin cytoskeleton networks in fission yeast imaged by spinning disk confocal microscopy. PMID:21822463

  20. Cytoskeletal proteins in cortical development and disease: actin associated proteins in periventricular heterotopia

    PubMed Central

    Lian, Gewei; Sheen, Volney L.

    2015-01-01

    The actin cytoskeleton regulates many important cellular processes in the brain, including cell division and proliferation, migration, and cytokinesis and differentiation. These developmental processes can be regulated through actin dependent vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. Many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes and proteins. Disruption in the actin cytoskeleton in the brain gives rise to periventricular heterotopia (PH), a malformation of cortical development, characterized by abnormal neurons clustered deep in the brain along the lateral ventricles. This disorder can give rise to seizures, dyslexia and psychiatric disturbances. Anatomically, PH is characterized by a smaller brain (impaired proliferation), heterotopia (impaired initial migration) and disruption along the neuroependymal lining (impaired cell-cell adhesion). Genes causal for PH have also been implicated in actin-dependent processes. The current review provides mechanistic insight into actin cytoskeletal regulation of cortical development in the context of this malformation of cortical development. PMID:25883548

  1. Cleavage furrow: timing of emergence of contractile ring actin filaments and establishment of the contractile ring by filament bundling in sea urchin eggs.

    PubMed

    Mabuchi, I

    1994-07-01

    Cleavage furrow formation at the first cell division of sea urchin and sand dollar eggs was investigated in detail by fluorescence staining of actin filaments with rhodamine-phalloidin of either whole eggs or isolated egg cortices. Cortical actin filaments were clustered at anaphase and then the clusters became fibrillar at the end of anaphase. The timing when the contractile ring actin filaments appear was precisely determined in the course of mitosis: accumulation of the contractile ring actin filaments at the equatorial cell cortex is first noticed at the beginning of telophase (shortly before furrow formation), when the chromosomal vesicles are fusing with each other. The accumulated actin filaments were not well organized at the early stage but were organized into parallel bundles as the furrowing progressed. The bundles were finally fused into a tightly packed filament belt. Wheat germ agglutinin (WGA)-binding sites were distributed on the surface of the egg in a manner similar to the actin filaments after anaphase. The WGA-binding sites became accumulated in the contractile ring together with the contractile ring actin filaments, indicating an intimate relationship between these sites and actin filament-anchoring sites on the plasma membrane. Myosin also appeared in the contractile ring together with the actin filaments. The 'cleavage stimulus', a signal hypothesized by Rappaport (reviewed by R. Rappaport (1986) Int. Rev. Cytol. 105, 245-281) was suggested to induce aggregation or bundling of the actin filaments in the cortical layer.

  2. Actinic Granuloma with Focal Segmental Glomerulosclerosis

    PubMed Central

    Phasukthaworn, Ruedee; Chanprapaph, Kumutnart; Vachiramon, Vasanop

    2016-01-01

    Actinic granuloma is an uncommon granulomatous disease, characterized by annular erythematous plaque with central clearing predominately located on sun-damaged skin. The pathogenesis is not well understood, ultraviolet radiation is recognized as precipitating factor. We report a case of a 52-year-old woman who presented with asymptomatic annular erythematous plaques on the forehead and both cheeks persisting for 2 years. The clinical presentation and histopathologic findings support the diagnosis of actinic granuloma. During that period of time, she also developed focal segmental glomerulosclerosis. The association between actinic granuloma and focal segmental glomerulosclerosis needs to be clarified by further studies. PMID:27293392

  3. Binding of actin to lens alpha crystallins

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

    Actin has been coupled to a cyanogen bromide-activated Sepharose 4B column, then tested for binding to alpha, beta, and gamma crystallin preparations from the bovine lens. Alpha, but not beta or gamma, crystallins bound to the actin affinity column in a time dependent and saturable manner. Subfractionation of the alpha crystallin preparation into the alpha-A and alpha-B species, followed by incubation with the affinity column, demonstrated that both species bound approximately the same. Together, these studies demonstrate a specific and saturable binding of lens alpha-A and alpha-B with actin.

  4. Dynamic reorganization of the actin cytoskeleton

    PubMed Central

    Gressin, Laurène; Théry, Manuel; Blanchoin, Laurent

    2015-01-01

    Cellular processes, including morphogenesis, polarization, and motility, rely on a variety of actin-based structures. Although the biochemical composition and filament organization of these structures are different, they often emerge from a common origin. This is possible because the actin structures are highly dynamic. Indeed, they assemble, grow, and disassemble in a time scale of a second to a minute. Therefore, the reorganization of a given actin structure can promote the formation of another. Here, we discuss such transitions and illustrate them with computer simulations. PMID:26989473

  5. Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules.

    PubMed

    Chun, Jong T; Limatola, Nunzia; Vasilev, Filip; Santella, Luigia

    2014-08-01

    We previously demonstrated that many aspects of the intracellular Ca(2+) increase in fertilized eggs of starfish are significantly influenced by the state of the actin cytoskeleton. In addition, the actin cytoskeleton appeared to play comprehensive roles in modulating cortical granules exocytosis and sperm entry during the early phase of fertilization. In the present communication, we have extended our work to sea urchin which is believed to have bifurcated from the common ancestor in the phylogenetic tree some 500 million years ago. To corroborate our earlier findings in starfish, we have tested how the early events of fertilization in sea urchin eggs are influenced by four different actin-binding drugs that promote either depolymerization or stabilization of actin filaments. We found that all the actin drugs commonly blocked sperm entry in high doses and significantly reduced the speed of the Ca(2+) wave. At low doses, however, cytochalasin B and phalloidin increased the rate of polyspermy. Overall, certain aspects of Ca(2+) signaling in these eggs were in line with the morphological changes induced by the actin drugs. That is, the time interval between the cortical flash and the first Ca(2+) spot at the sperm interaction site (the latent period) was significantly prolonged in the eggs pretreated with cytochalasin B or latrunculin A, whereas the Ca(2+) decay kinetics after the peak was specifically attenuated in the eggs pretreated with jasplakinolide or phalloidin. In addition, the sperm interacting with the eggs pretreated with actin drugs often generated multiple Ca(2+) waves, but tended to fail to enter the egg. Thus, our results indicated that generation of massive Ca(2+) waves is neither indicative of sperm entry nor sufficient for cortical granules exocytosis in the inseminated sea urchin eggs, whereas the structure and functionality of the actin cytoskeleton are the major determining factors in the two processes.

  6. Actin-binding protein (ABP-280) filamin gene (FLN) maps telomeric to the color vision locus (R/GCP) and centromeric to G6PD in Xq28

    SciTech Connect

    Gorlin, J.B. Dana-Farber Cancer Institute, Boston, MA ); Henske, E.; Hartwig, J.H.; Kwiatkowski, D.J. ); Warren, S.T.; Kunst, C.B. ); D'Urso, M.; Palmieri, G. ); Bruns, G. )

    1993-08-01

    Actin-binding protein-280 (ABP-280) is a dimeric actin filament-crosslinking protein that promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The authors have mapped the ABP-280 filamin gene (FLN) to Xq28 by Southern blot analysis of somatic cell hybrid lines, by fluorescence in situ hybridization, and through identification of portions of the FLN gene within cosmids and YACs mapped to Xq28. The FLN gene is found within a 200-kb region centromeric to the G6PD locus and telomeric to DSX52 and the color vision locus. 23 refs., 2 figs.

  7. Actin bundling by dynamin 2 and cortactin is implicated in cell migration by stabilizing filopodia in human non-small cell lung carcinoma cells

    PubMed Central

    Yamada, Hiroshi; Takeda, Tetsuya; Michiue, Hiroyuki; Abe, Tadashi; Takei, Kohji

    2016-01-01

    The endocytic protein dynamin participates in the formation of actin-based membrane protrusions such as podosomes, pseudopodia, and invadopodia, which facilitate cancer cell migration, invasion, and metastasis. However, the role of dynamin in the formation of actin-based membrane protrusions at the leading edge of cancer cells is unclear. In this study, we demonstrate that the ubiquitously expressed dynamin 2 isoform facilitates cell migration by stabilizing F-actin bundles in filopodia of the lung cancer cell line H1299. Pharmacological inhibition of dynamin 2 decreased cell migration and filopodial formation. Furthermore, dynamin 2 and cortactin mostly colocalized along F-actin bundles in filopodia of serum-stimulated H1299 cells by immunofluorescent and immunoelectron microscopy. Knockdown of dynamin 2 or cortactin inhibited the formation of filopodia in serum-stimulated H1299 cells, concomitant with a loss of F-actin bundles. Expression of wild-type cortactin rescued the punctate-like localization of dynamin 2 and filopodial formation. The incubation of dynamin 2 and cortactin with F-actin induced the formation of long and thick actin bundles, with these proteins colocalizing at F-actin bundles. A depolymerization assay revealed that dynamin 2 and cortactin increased the stability of F-actin bundles. These results indicate that dynamin 2 and cortactin participate in cell migration by stabilizing F-actin bundles in filopodia. Taken together, these findings suggest that dynamin might be a possible molecular target for anticancer therapy. PMID:27572123

  8. Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

    The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

  9. Nanoscopy of filamentous actin in cortical dendrites of a living mouse.

    PubMed

    Willig, Katrin I; Steffens, Heinz; Gregor, Carola; Herholt, Alexander; Rossner, Moritz J; Hell, Stefan W

    2014-01-01

    We demonstrate superresolution fluorescence microscopy (nanoscopy) of protein distributions in a mammalian brain in vivo. Stimulated emission depletion microscopy reveals the morphology of the filamentous actin in dendritic spines down to 40 μm in the molecular layer of the visual cortex of an anesthetized mouse. Consecutive recordings at 43-70 nm resolution reveal dynamical changes in spine morphology.

  10. Correlation between polymerizability and conformation in scallop beta-like actin and rabbit skeletal muscle alpha-actin.

    PubMed

    Khaitlina, S; Antropova, O; Kuznetsova, I; Turoverov, K; Collins, J H

    1999-08-01

    In order to investigate the structural basis for functional differences among actin isoforms, we have compared the polymerization properties and conformations of scallop adductor muscle beta-like actin and rabbit skeletal muscle alpha-actin. Polymerization of scallop Ca(2+)-actin was slower than that of skeletal muscle Ca(2+)-actin. Cleavage of the actin polypeptide chain between Gly-42 and Val-43 with Escherichia coli protease ECP 32 impaired the polymerization of scallop Mg(2+)-actin to a greater extent than skeletal muscle Mg(2+)-actin. When monomeric scallop and skeletal muscle Ca(2+)-actins were subjected to limited proteolysis with trypsin, subtilisin, or ECP 32, no differences in the conformation of actin subdomain 2 were detected. At the same time, local differences in the conformations of scallop and skeletal muscle actin subdomains 1 were revealed as intrinsic fluorescence differences. Replacement of tightly bound Ca(2+) with Mg(2+) resulted in more extensive proteolysis of segment 61-69 of scallop actin than in the case of skeletal muscle actin. Furthermore, segment 61-69 was more accessible to proteolysis with subtilisin in polymerized scallop Ca(2+)-actin than in polymerized skeletal muscle Ca(2+)-actin, indicating that, in the polymeric form, the nucleotide-containing cleft is in a more open conformation in beta-like scallop actin than in skeletal muscle alpha-actin. We suggest that this difference between scallop and skeletal muscle actins is due to a less efficient shift of scallop actin subdomain 2 to the position it has in the polymer. The possible consequences of amino acid substitutions in actin subdomain 1 in the allosteric regulation of the actin cleft, and hence in the different stabilities of polymers formed by different actins, are discussed. PMID:10415117

  11. Structural Differences Explain Diverse Functions of Plasmodium Actins

    PubMed Central

    Vahokoski, Juha; Martinez, Silvia Muñico; Ignatev, Alexander; Lepper, Simone; Frischknecht, Friedrich; Sidén-Kiamos, Inga; Sachse, Carsten; Kursula, Inari

    2014-01-01

    Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties. PMID:24743229

  12. Actin filament organization of foot processes in vertebrate glomerular podocytes.

    PubMed

    Ichimura, Koichiro; Kurihara, Hidetake; Sakai, Tatsuo

    2007-09-01

    We investigated the actin filament organization and immunolocalization of actin-binding proteins (alpha-actinin and cortactin) in the podocyte foot processes of eight vertebrate species (lamprey, carp, newt, frog, gecko, turtle, quail, and rat). Three types of actin cytoskeleton were found in these foot processes. (1) A cortical actin network with cortactin filling the space between the plasma membrane and the other actin cytoskeletons described below was found in all of the species examined here. The data indicated that the cortical actin network was the minimal essential actin cytoskeleton for the formation and maintenance of the foot processes in vertebrate podocytes. (2) An actin bundle with alpha-actinin existing along the longitudinal axis of foot process above the level of slit diaphragms was only observed in quail and rat. (3) An actin fascicle consisting of much fewer numbers of actin filaments than that of the actin bundle was observed in the species other than quail and rat, but at various frequencies. These findings suggest that the actin bundle is an additional actin cytoskeleton reflecting a functional state peculiar to quail and rat glomeruli. Considering the higher intraglomerular pressure and the extremely thin filtration barrier in birds and mammals, the foot processes probably mainly protect the thinner filtration barrier from the higher internal pressure occurring in quail and rat glomeruli. Therefore, we consider that the actin bundle plays a crucial role in the mechanical protection of the filtration barrier. Moreover, the actin fascicle may be a potential precursor of the actin bundle.

  13. Genetics Home Reference: actin-accumulation myopathy

    MedlinePlus

    ... 7(3):160-8. Citation on PubMed Laing NG, Dye DE, Wallgren-Pettersson C, Richard G, Monnier ... Vigneron J, Wallgren-Pettersson C, Beggs AH, Laing NG. Mutations in the skeletal muscle alpha-actin gene ...

  14. [Actin in the wound healing process].

    PubMed

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

    2009-01-01

    Wound healing is an important biological process of crucial value for organisms survival and retention of its proper functions. The recognition of molecular mechanisms of these phenomenon is still under investigation. The transition of mesenchymal fibroblasts to myofibroblasts is a key point in wound healing. The contraction ability of myofibroblast enables the shrinkage of a wound and closes its edges. Alpha smooth muscle actin (alpha-SMA), one of six actin isoforms, is a marker of compeletely differentiated myofibroblast. The regulation of differentiation process depends on many growth factors (especially TGF beta 1), the level of active thymosin beta 4, extracellular matrix proteins--including fibronectin, and also on specificity of microenvironment. Thymosin beta 4 is responsible for maintenance of pool of monomeric actin and actin filaments depolymerization. It can also act as a transcription factor, migration stimulator and immunomodulator, so this protein deserves for more attention in wound healing research field. PMID:19824469

  15. Structural Dynamics of an Actin Spring

    PubMed Central

    Mahadevan, L.; Riera, C.S.; Shin, Jennifer H.

    2011-01-01

    Actin-based motility in cells is usually associated with either polymerization/depolymerization in the presence of cross-linkers or contractility in the presence of myosin motors. Here, we focus on a third distinct mechanism involving actin in motility, seen in the dynamics of an active actin spring that powers the acrosomal reaction of the horseshoe crab (Limulus polyphemus) sperm. During this process, a 60-μm bent and twisted bundle of cross-linked actin uncoils and becomes straight in a few seconds in the presence of Ca2+. This straightening, which occurs at a constant velocity, allows the acrosome to forcefully penetrate the egg. Synthesizing ultrastructural information with the kinetics, energetics, and imaging of calcium binding allows us to construct a dynamical theory for this mechanochemical engine consistent with our experimental observations. It also illuminates the general mechanism by which energy may be stored in conformational changes and released cooperatively in ordered macromolecular assemblies. PMID:21320427

  16. Mechanics model for actin-based motility.

    PubMed

    Lin, Yuan

    2009-02-01

    We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.

  17. Mechanics model for actin-based motility

    NASA Astrophysics Data System (ADS)

    Lin, Yuan

    2009-02-01

    We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.

  18. Actinic review of EUV masks

    NASA Astrophysics Data System (ADS)

    Feldmann, Heiko; Ruoff, Johannes; Harnisch, Wolfgang; Kaiser, Winfried

    2010-04-01

    Management of mask defects is a major challenge for the introduction of EUV for HVM production. Once a defect has been detected, its printing impact needs to be predicted. Potentially the defect requires some repair, the success of which needs to be proven. This defect review has to be done with an actinic inspection system that matches the imaging conditions of an EUV scanner. During recent years, several concepts for such an aerial image metrology system (AIMS™) have been proposed. However, until now no commercial solution exists for EUV. Today, advances in EUV optics technology allow envisioning a solution that has been discarded before as unrealistic. We present this concept and its technical cornerstones.While the power requirement for the EUV source is less demanding than for HVM lithography tools, radiance, floor space, and stability are the main criteria for source selection. The requirement to emulate several generations of EUV scanners demands a large flexibility for the ilumination and imaging systems. New critical specifications to the EUV mirrors in the projection microscope can be satisfied using our expertise from lithographic mirrors. In summary, an EUV AIMS™ meeting production requirements seems to be feasible.

  19. Elasticity of F-actin networks

    NASA Astrophysics Data System (ADS)

    Gardel, Margaret Lise

    This thesis presents a study of the elasticity and microstructure of three filamentous actin (F-actin) based materials. Using bulk rheology, microrheology, multiple particle tracking and imaging techniques, we study the microscopic origins of the mechanical properties of F-actin networks. We briefly introduce aspects of F-actin and rheology essential to provide a background for and motivate this thesis in Chapter 1. In Chapter 2, we describe the materials and methods used. An introduction to microrheology is given in Chapter 3. In Chapter 4, we study solutions of entangled F-actin. We elucidate the microscopic origins of bulk elasticity using microrheology techniques. We also show that multiple particle tracking can also probe the dynamics of the F-actin solution microstructure. We explore the effect of rigid, incompliant chemical cross-links between actin filaments in Chapter 5. We explore changes in the network microstructure as the concentration of cross-links is varied. We find that the elastic stiffness of these networks is extremely sensitive to small changes in cross-link density. Despite this large variation, the linear viscoelasticity of all networks can be scaled onto a universal master curve; this scaling reveals that the mechanical dissipation of the networks is due to thermal fluctuations of F-actin. At large stresses, the mechanical stiffness of these networks diverges. The form of this stress stiffening response is consistent with the non-linear force extension of a single semi-flexible polymer. Thus, over a large range of conditions, the linear and nonlinear mechanical response of rigidly cross-linked networks is entropic in origin. Finally, at very low cross-link and filament densities, we observe a transition to a qualitatively different type of elasticity; this is consistent with a transition to an enthalpic network elasticity dominated by bending of F-actin. In Chapter 6, we study the elastic properties of F-actin networks assembled with a

  20. Mechanism of Actin Filament Bundling by Fascin

    SciTech Connect

    Jansen, Silvia; Collins, Agnieszka; Yang, Changsong; Rebowski, Grzegorz; Svitkina, Tatyana; Dominguez, Roberto

    2013-03-07

    Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four {beta}-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in {beta}-trefoil domains 1 and 3. The site in {beta}-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in {beta}-trefoil-3 is related by pseudo-2-fold symmetry to that in {beta}-trefoil-1. The two sites are {approx}5 nm apart, resulting in a distance between actin filaments in the bundle of {approx}8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

  1. Actin: its cumbersome pilgrimage through cellular compartments.

    PubMed

    Schleicher, Michael; Jockusch, Brigitte M

    2008-06-01

    In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days' knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin.

  2. Sarcomeric Pattern Formation by Actin Cluster Coalescence

    PubMed Central

    Friedrich, Benjamin M.; Fischer-Friedrich, Elisabeth; Gov, Nir S.; Safran, Samuel A.

    2012-01-01

    Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells. PMID:22685394

  3. Kinetic multi-layer model of the epithelial lining fluid (KM-ELF): Reactions of ozone and OH with antioxidants and surfactant molecules

    NASA Astrophysics Data System (ADS)

    Lakey, Pascale; Pöschl, Ulrich; Shiraiwa, Manabu

    2015-04-01

    Oxidants cause damage to biosurfaces such as the lung epithelium unless they are effectively scavenged. The respiratory tract is covered in a thin layer of fluid which extends from the nasal cavity to the alveoli and contain species that scavenge ozone and other incoming oxidants. The kinetic multi-layer model of the epithelial lining fluid (KM-ELF) has been developed in order to investigate the reactions of ozone and OH with antioxidants (ascorbate, uric acid, glutathione and α-tocopherol) and surfactant lipids and proteins within the epithelial lining fluid (ELF). The model incorporates different processes: gas phase diffusion, adsorption and desorption from the surface, bulk phase diffusion and known reactions at the surface and in the bulk. The ELF is split into many layers: a sorption layer, a surfactant layer, a near surface bulk layer and several bulk layers. Initial results using KM-ELF indicate that at ELF thicknesses of 80 nm and 1 × 10-4cm the ELF would become rapidly saturated with ozone with saturation occurring in less than a second. However, at an ELF thickness of 1 × 10-3cm concentration gradients were observed throughout the ELF and the presence of antioxidants reduced the O3 reaching the lung cells and tissues by 40% after 1 hour of exposure. In contrast, the antioxidants were efficient scavengers of OH radicals, although the large rate constants of OH reacting with the antioxidants resulted in the antioxidants decaying away rapidly. The chemical half-lives of the antioxidants and surface species were also calculated using KM-ELF as a function of O3 and OH concentration and ELF thickness. Finally, the pH dependence of the products of reactions between antioxidants and O3 were investigated. The KM-ELF model predicted that a harmful ascorbate ozonide product would increase from 1.4 × 1011cm-3at pH 7.4 to 1.1 × 1014 cm-3 at pH 4after 1 hour although a uric acid ozonide product would decrease from 2.0 × 1015cm-3to 5.9 × 1012cm-3.

  4. Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

    NASA Astrophysics Data System (ADS)

    Janmey, Paul A.; Hvidt, Søren; Lamb, Jennifer; Stossel, Thomas P.

    1990-05-01

    THE maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex1,2. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel sol' transformations result from the rearrangement of cortical actin-rich networks3. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, α-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments4: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels Theologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.

  5. Nuclear and cytoplasmic actin in dinoflagellates.

    PubMed

    Soyer-Gobillard, M O; Ausseil, J; Géraud, M L

    1996-01-01

    Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the

  6. Interactions of actin, myosin, and an actin-binding protein of chronic myelogenous leukemia leukocytes.

    PubMed Central

    Boxer, L A; Stossel, T P

    1976-01-01

    Actin, myosin, and a high molecular weight actin-binding protein were purified from chronic myelogenous leukemia (CML) leukocytes. CML leukocyte actin resembled skeletal muscle and other cytoplasmic actins by its subunit molecular weight, by its ability to polymerize in the presence of salts, and to activate the Mg2+-ATPase activity of rabbit skeletal muscle myosin. CML leukocyte myosin was similar to other vertebrate cytoplasmic myosins in having heavy chains and two light subunits. However, its apparent heavy-chain molecular weight and Stokes radius suggested that it was variably degraded during purification. Purified CML leukocyte myosin had average specific EDTA- AND Ca2+-activated ATPase activities of 125 and 151 nmol Pi released/mg protein per min, respectively and low specific Mg2+-ATPase activity. The Mg2+-ATPase activity of CML myosin was increased 200-fold by rabbit skeletal muscle F-actin, but the specific activity relative to that of actin-activated rabbit skeletal muscle myosin was low. CML leukocyte myosin, like other vertebrate cytoplasmic myosins, formed filaments in 0.1 M KCl solutions. Reduced and denatured CML leukocyte-actin-binding protein had a single high molecular weight subunit like a recently described actin-binding protein of rabbit pulmonary macrophages which promotes the polymerization and gelation of actin. Cytoplasmic extracts of CML leukocytes prepared with ice-cold 0.34-M sucrose solutions containing Mg2+-ATP, dithiothreitol, and EDTA at pH 7.0 underwent rapid gelation when warmed to 25 degrees C. Initially, the gel could be liquified by cooling to ice-bath temperature. With time, warmed cytoplasmic extract gels shrunk ("contracted") into aggregates. The following findings indicated that CML leukocyte actin-binding protein promoted the temperature-dependent gelation of actin in the cytoplasmic extracts and that CML leukocyte myosin was involved in the contraction of the actin gels: (a) Cytoplasmic extract gels initially contained

  7. Protein phosphatase 2A, a potential regulator of actin dynamics and actin-based organelle motility in the green alga Acetabularia.

    PubMed

    Menzel, D; Vugrek, O; Frank, S; Elsner-Menzel, C

    1995-06-01

    The giant, unicellular alga Acetabularia is a well known experimental model for the study of actin-dependent intracellular organelle motility. In the cyst stage, however, which is equivalent to the gametophytic stage, organelles are immobile, even though an actin cytoskeleton is present. The reason for the lack of organelle motility at this stage has not been known. To test the hypothesis that organelle motility could be under the control of posttranslational modification by protein phosphorylation, we have treated cysts with submicromolar concentrations of okadaic acid or calyculin A, both potent inhibitors of serine/threonine protein phosphatases (ser/thr-PPases). The effects were dramatic: Instead of linear actin bundles typical for control cysts, circular arrays of actin bundles formed in the cortical cyst cytoplasm. Concomitant with the formation of these action rings, the cytoplasmic layers beneath the rings began to slowly rotate in a continuous and uniform counter-clockwise fashion. This effect suggests that protein phosphorylation acts on the actin cytoskeleton at two levels: (1) It changes the assembly properties of the actin filament system to the extent that novel cytoskeletal configurations are formed and (2) it raises the activity of putative motor proteins involved in the rotational movements to levels sufficiently high to support motility at a stage when organelle motility does not normally occur. Northern blot analysis of cyst stage-mRNA using probes specific to protein phosphatase type 1 (PP1) and type 2A (PP2A) reveals that PP2A is strongly expressed at this developmental stage whereas PP1 is not detectable, suggesting that PP2A is the likely target to the protein phosphatase inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Defining a core set of actin cytoskeletal proteins critical for actin-based motility of Rickettsia.

    PubMed

    Serio, Alisa W; Jeng, Robert L; Haglund, Cat M; Reed, Shawna C; Welch, Matthew D

    2010-05-20

    Many Rickettsia species are intracellular bacterial pathogens that use actin-based motility for spread during infection. However, while other bacteria assemble actin tails consisting of branched networks, Rickettsia assemble long parallel actin bundles, suggesting the use of a distinct mechanism for exploiting actin. To identify the underlying mechanisms and host factors involved in Rickettsia parkeri actin-based motility, we performed an RNAi screen targeting 115 actin cytoskeletal genes in Drosophila cells. The screen delineated a set of four core proteins-profilin, fimbrin/T-plastin, capping protein, and cofilin--as crucial for determining actin tail length, organizing filament architecture, and enabling motility. In mammalian cells, these proteins were localized throughout R. parkeri tails, consistent with a role in motility. Profilin and fimbrin/T-plastin were critical for the motility of R. parkeri but not Listeria monocytogenes. Our results highlight key distinctions between the evolutionary strategies and molecular mechanisms employed by bacterial pathogens to assemble and organize actin. PMID:20478540

  9. SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics.

    PubMed

    Hung, Ruei-Jiun; Spaeth, Christopher S; Yesilyurt, Hunkar Gizem; Terman, Jonathan R

    2013-12-01

    Actin's polymerization properties are markedly altered by oxidation of its conserved Met 44 residue. Mediating this effect is a specific oxidation-reduction (redox) enzyme, Mical, that works with Semaphorin repulsive guidance cues and selectively oxidizes Met 44. We now find that this actin-regulatory process is reversible. Employing a genetic approach, we identified a specific methionine sulfoxide reductase (MsrB) enzyme SelR that opposes Mical redox activity and Semaphorin-Plexin repulsion to direct multiple actin-dependent cellular behaviours in vivo. SelR specifically catalyses the reduction of the R isomer of methionine sulfoxide (methionine-R-sulfoxide) to methionine, and we found that SelR directly reduced Mical-oxidized actin, restoring its normal polymerization properties. These results indicate that Mical oxidizes actin stereospecifically to generate actin Met-44-R-sulfoxide (actin(Met(R)O-44)), and also implicate the interconversion of specific Met/Met(R)O residues as a precise means to modulate protein function. Our results therefore uncover a specific reversible redox actin regulatory system that controls cell and developmental biology.

  10. Size-Induced Depression of First-Order Transition Lines and Entropy Jump in Extremely Layered Nanocrystalline Vortex Matter

    NASA Astrophysics Data System (ADS)

    Dolz, M. I.; Fasano, Y.; Cejas Bolecek, N. R.; Pastoriza, H.; Mosser, V.; Li, M.; Konczykowski, M.

    2015-09-01

    We detect the persistence of the solidification and order-disorder first-order transition lines in the phase diagram of nanocrystalline Bi2 Sr2 CaCu2 O8 vortex matter down to a system size of less than one hundred vortices. The temperature location of the vortex solidification transition line is not altered by decreasing the sample size although there is a depletion of the entropy jump at the transition with respect to macroscopic vortex matter. The solid order-disorder phase transition field moves upward on decreasing the system size due to the increase of the surface-to-volume ratio of vortices entailing a decrease on the average vortex binding energy.

  11. Characterization of the activities of actin-affecting drugs on tumor cell migration

    SciTech Connect

    Hayot, Caroline; Debeir, Olivier; Ham, Philippe van; Damme, Marc van; Kiss, Robert; Decaestecker, Christine . E-mail: cdecaes@ulb.ac.be

    2006-02-15

    Metastases kill 90% of cancer patients. It is thus a major challenge in cancer therapy to inhibit the spreading of tumor cells from primary tumor sites to those particular organs where metastases are likely to occur. Whereas the actin cytoskeleton is a key component involved in cell migration, agents targeting actin dynamics have been relatively poorly investigated. Consequently, valuable in vitro pharmacological tools are needed to selectively identify this type of agent. In response to the absence of any standardized process, the present work aims to develop a multi-assay strategy for screening actin-affecting drugs with anti-migratory potentials. To validate our approach, we used two cancer cell lines (MCF7 and A549) and three actin-affecting drugs (cytochalasin D, latrunculin A, and jasplakinolide). We quantified the effects of these drugs on the kinetics of actin polymerization in tubes (by means of spectrofluorimetry) and on the dynamics of actin cytoskeletons within whole cells (by means of fluorescence microscopy). Using quantitative videomicroscopy, we investigated the actual effects of the drugs on cell motility. Finally, the combined drug effects on cell motility and cell growth were evaluated by means of a scratch-wound assay. While our results showed concordant drug-induced effects on actin polymerization occurring in vitro in test tubes and within whole cells, the whole cell assay appeared more sensitive than the tube assay. The inhibition of actin polymerization induced by cytochalasin D was paralleled by a decrease in cell motility for both cell types. In the case of jasplakinolide, which induces actin polymerization, while it significantly enhanced the locomotion of the A549 cells, it significantly inhibited that of the MCF-7 ones. All these effects were confirmed by means of the scratch-wound assay except of the jasplakinolide-induced effects on MCF-7 cell motility. These later seemed compensated by an additional effect occurring during wound

  12. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.

    PubMed

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

  13. Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles.

    PubMed

    Mavrakis, Manos; Azou-Gros, Yannick; Tsai, Feng-Ching; Alvarado, José; Bertin, Aurélie; Iv, Francois; Kress, Alla; Brasselet, Sophie; Koenderink, Gijsje H; Lecuit, Thomas

    2014-04-01

    Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

  14. Design, synthesis, and biological evaluation of simplified side chain hybrids of the potent actin binding polyketides rhizopodin and bistramide.

    PubMed

    Herkommer, Daniel; Dreisigacker, Sandra; Sergeev, Galina; Sasse, Florenz; Gohlke, Holger; Menche, Dirk

    2015-03-01

    The natural products rhizopodin and bistramide belong to an elite class of highly potent actin binding agents. They show powerful antiproliferative activities against a range of tumor cell lines, with IC50 values in the low-nanomolar range. At the molecular level they disrupt the actin cytoskeleton by binding specifically to a few critical sites of G-actin, resulting in actin filament stabilization. The important biological properties of rhizopodin and bistramide, coupled with their unique and intriguing molecular architectures, render them attractive compounds for further development. However, this is severely hampered by the structural complexity of these metabolites. We initiated an interdisciplinary approach at the interface between molecular modeling, organic synthesis, and chemical biology to support further biological applications. We also wanted to expand structure-activity relationship studies with the goal of accessing simplified analogues with potent biological properties. We report computational analyses of actin-inhibitor interactions involving molecular docking, validated on known actin binding ligands, that show a close match between the crystal and modeled structures. Based on these results, the ligand shape was simplified, and more readily accessible rhizopodin-bistramide mimetics were designed. A flexible and modular strategy was applied for the synthesis of these compounds, enabling diverse access to dramatically simplified rhizopodin-bistramide hybrids. This novel analogue class was analyzed for its antiproliferative and actin binding properties. PMID:25641798

  15. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth[W

    PubMed Central

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-01-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes. PMID:24058157

  16. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    SciTech Connect

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  17. Purification and Characterization of Actin from Maize Pollen 1

    PubMed Central

    Liu, Xiong; Yen, Lung-Fei

    1992-01-01

    Pollen is an excellent source of actin for biochemical and physiological studies of the actomyosin system in higher plants. We have developed an efficient method to prepare relatively high levels of actin from the pollen of maize (Zea mays L.). The procedures of purification include acetone powder preparation, saturated ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, a cycle of polymerization-depolymerization, and Sephacryl S-200 gel filtration. The average yield of actin is 19 milligrams per 100 grams of pollen grains extracted. This is comparable with those of Acanthamoeba castellanii and human platelets. The purified pollen actin is electrophoretically homogeneous and its molecular mass is 42 kilodaltons. The amino acid composition and circular dichroism spectrum of pollen actin are identical to those of muscle actin. The actin purified from pollen is able to polymerize to F-actin. The pollen F-actin activated the activity of the muscle myosin ATPase sevenfold. ImagesFigure 1Figure 2 PMID:16668982

  18. Tau co-organizes dynamic microtubule and actin networks

    PubMed Central

    Elie, Auréliane; Prezel, Elea; Guérin, Christophe; Denarier, Eric; Ramirez-Rios, Sacnicte; Serre, Laurence; Andrieux, Annie; Fourest-Lieuvin, Anne; Blanchoin, Laurent; Arnal, Isabelle

    2015-01-01

    The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts. PMID:25944224

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

    SciTech Connect

    Hirano, Hidemi; Matsuura, Yoshiyuki

    2011-10-22

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

  20. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    PubMed Central

    Paves, Heiti; Truve, Erkki

    2004-01-01

    Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area. PMID:15102327

  1. The EGF receptor is an actin-binding protein

    PubMed Central

    1992-01-01

    In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996. PMID:1383230

  2. Crystal structure of a nuclear actin ternary complex.

    PubMed

    Cao, Tingting; Sun, Lingfei; Jiang, Yuxiang; Huang, Shanjin; Wang, Jiawei; Chen, Zhucheng

    2016-08-01

    Actin polymerizes and forms filamentous structures (F-actin) in the cytoplasm of eukaryotic cells. It also exists in the nucleus and regulates various nucleic acid transactions, particularly through its incorporation into multiple chromatin-remodeling complexes. However, the specific structure of actin and the mechanisms that regulate its polymeric nature inside the nucleus remain unknown. Here, we report the crystal structure of nuclear actin (N-actin) complexed with actin-related protein 4 (Arp4) and the helicase-SANT-associated (HSA) domain of the chromatin remodeler Swr1. The inner face and barbed end of N-actin are sequestered by interactions with Arp4 and the HSA domain, respectively, which prevents N-actin from polymerization and binding to many actin regulators. The two major domains of N-actin are more twisted than those of globular actin (G-actin), and its nucleotide-binding pocket is occluded, freeing N-actin from binding to and regulation by ATP. These findings revealed the salient structural features of N-actin that distinguish it from its cytoplasmic counterpart and provide a rational basis for its functions and regulation inside the nucleus. PMID:27457955

  3. The actinome of Dictyostelium discoideum in comparison to actins and actin-related proteins from other organisms.

    PubMed

    Joseph, Jayabalan M; Fey, Petra; Ramalingam, Nagendran; Liu, Xiao I; Rohlfs, Meino; Noegel, Angelika A; Müller-Taubenberger, Annette; Glöckner, Gernot; Schleicher, Michael

    2008-07-09

    Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.

  4. Analysis of the substrate influence on the ordering of epitaxial molecular layers: The special case of point-on-line coincidence

    NASA Astrophysics Data System (ADS)

    Mannsfeld, S. C.; Fritz, T.

    2004-02-01

    The physical structure of organic-inorganic heteroepitaxial thin films is usually governed by a fine balance between weak molecule-molecule interactions and a weakly laterally varying molecule-substrate interaction potential. Therefore, in order to investigate the energetics of such a layer system one has to consider large molecular domains. So far, layer potential calculations for large domains of organic thin films on crystalline substrates were difficult to perform concerning the computational effort which stems from the vast number of atoms which have to be included. Here, we present a technique which enables the calculation of the molecule-substrate interaction potential for large molecular domains by utilizing potential energy grid files. This technique allows the investigation of the substrate influence in systems prepared by organic molecular beam epitaxy (OMBE), like 3,4,9,10-perylenetetracarboxylicdianhydride on highly oriented pyrolytic graphite. For this system the so-called point-on-line coincidence was proposed, a growth mode which has been controversially discussed in literature. Furthermore, we are able to provide evidence for a general energetic advantage of such point-on-line coincident domain orientations over arbitrarily oriented domains which substantiates that energetically favorable lattice structures in OMBE systems are not restricted to commensurate unit cells or coincident super cells.

  5. Impact of Carbon Nanomaterials on Actin Polymerization.

    PubMed

    Dong, Ying; Sun, Haiyan; Li, Xu; Li, Xin; Zhao, Lina

    2016-03-01

    Many nanomaterials have entered people's daily lives and impact the normal process of biological entities consequently. As one kind of the important nanomaterials, carbon based nanomaterials have invoked a lot of concerns from scientific researches because of their unique physicochemical properties. In eukaryotes, actin is the most abundantly distributed protein in both cytoplasm and cell nucleus, and closely controls the cell proliferation and mobility. Recently, many investigations have found some carbon based nanomaterials can affect actin cytoskeleton remarkably, including fullerenes derivatives, carbon nanotubes, graphene and its derivatives. However, these interaction processes are complicated and the underlying mechanism is far from being understood clearly. In this review, we discussed the different mechanisms of carbon nanomaterials impact on actin polymerization into three pathways, as triggering the signaling pathways from carbon nanomaterials outside of cells, increasing the production of reactive oxygen species from carbon nanomaterials inside of cells and direct interaction from carbon nanomaterials inside of cells. As a result, the dimension and size of carbon nanomaterials play a key role in regulation of actin cytoskeleton. Furthermore, we forecasted the possible investigation strategy for meeting the challenges of the future study on this topic. We hope the findings are helpful in understanding the molecular mechanism in carbon nanomaterials regulating actin polymerization, and provide new insight in novel nanomedicine development for inhibition tumor cell migration. PMID:27455649

  6. The carboxyl terminus of the alpha-subunit of the amiloride-sensitive epithelial sodium channel binds to F-actin.

    PubMed

    Mazzochi, Christopher; Bubien, James K; Smith, Peter R; Benos, Dale J

    2006-03-10

    The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC. PMID:16356937

  7. Geldanamycin anisimycins activate Rho and stimulate Rho- and ROCK-dependent actin stress fiber formation.

    PubMed

    Amiri, Anahita; Noei, Farahnaz; Feroz, Tahir; Lee, Jonathan M

    2007-09-01

    Heat shock protein 90 (Hsp90) is a member of the heat shock family of molecular chaperones that regulate protein conformation and activity. Hsp90 regulates multiple cell signaling pathways by controlling the abundance and activity of several important protein kinases and cell cycle-related proteins. In this report, we show that inhibition of Hsp90 by geldanamycin or its derivative, 17-allylamino-17-desmethoxygeldamycin, leads to activation of the Rho GTPase and a dramatic increase in actin stress fiber formation in human tumor cell lines. Inactivation of Rho prevents geldanamycin-induced actin reorganization. Hsp90 inactivation does not alter the appearance of filopodia or lamellipodia and tubulin architecture is not visibly perturbed. Our observations suggest that Hsp90 has an important and specific role in regulating Rho activity and Rho-dependent actin cytoskeleton remodeling.

  8. Shielding of the Geomagnetic Field Alters Actin Assembly and Inhibits Cell Motility in Human Neuroblastoma Cells

    PubMed Central

    Mo, Wei-Chuan; Zhang, Zi-Jian; Wang, Dong-Liang; Liu, Ying; Bartlett, Perry F.; He, Rong-Qiao

    2016-01-01

    Accumulating evidence has shown that absence of the geomagnetic field (GMF), the so-called hypomagnetic field (HMF) environment, alters the biological functions in seemingly non-magnetosensitive cells and organisms, which indicates that the GMF could be sensed by non-iron-rich and non-photo-sensing cells. The underlying mechanisms of the HMF effects on those cells are closely related to their GMF sensation but remain poorly understood so far. Previously, we found that the HMF represses expressions of genes associated with cell migration and cytoskeleton assembly in human neuroblastoma cells (SH-SY5Y cell line). Here, we measured the HMF-induced changes on cell morphology, adhesion, motility and actin cytoskeleton in SH-SY5Y cells. The HMF inhibited cell adhesion and migration accompanied with a reduction in cellular F-actin amount. Moreover, following exposure to the HMF, the number of cell processes was reduced and cells were smaller in size and more round in shape. Furthermore, disordered kinetics of actin assembly in vitro were observed during exposure to the HMF, as evidenced by the presence of granule and meshed products. These results indicate that elimination of the GMF affects assembly of the motility-related actin cytoskeleton, and suggest that F-actin is a target of HMF exposure and probably a mediator of GMF sensation. PMID:27029216

  9. A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

    PubMed

    Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

    2013-04-01

    The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

  10. Structural Transitions of F-Actin:Espin Bundles

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin; Bartles, James; Wong, Gerard

    2006-03-01

    Espin is an actin bundling protein involved in the formation of the parallel bundles of filamentous actin in hair cell stereocilia. Mutations in espin are implicated in deafness phenotypes in mice and humans. We present measurements of the F-actin structures induced by wild type and by mutated espin obtained via small angle x-ray scattering and fluorescence microscopy. We found that wild type espin induced a paracrystalline hexagonal array of twisted F-actin, whereas the mutated espin only condensed the F-actin into a nematic-like phase. The possibility of coexisting nematic and bundled actin in mixtures containing both mutant and wild type espins was also investigated.

  11. Nuclear actin and lamins in viral infections.

    PubMed

    Cibulka, Jakub; Fraiberk, Martin; Forstova, Jitka

    2012-03-01

    Lamins are the best characterized cytoskeletal components of the cell nucleus that help to maintain the nuclear shape and participate in diverse nuclear processes including replication or transcription. Nuclear actin is now widely accepted to be another cytoskeletal protein present in the nucleus that fulfills important functions in the gene expression. Some viruses replicating in the nucleus evolved the ability to interact with and probably utilize nuclear actin for their replication, e.g., for the assembly and transport of capsids or mRNA export. On the other hand, lamins play a role in the propagation of other viruses since nuclear lamina may represent a barrier for virions entering or escaping the nucleus. This review will summarize the current knowledge about the roles of nuclear actin and lamins in viral infections.

  12. Actin Filament Segmentation Using Dynamic Programming

    PubMed Central

    Li, Hongsheng; Shen, Tian; Huang, Xiaolei

    2011-01-01

    We introduce a novel algorithm for actin filament segmentation in 2D TIRFM image sequences. This problem is difficult because actin filaments dynamically change shapes during their growth, and the TIRFM images are usually noisy. We ask a user to specify the two tips of a filament of interest in the first frame. We then model the segmentation problem in an image sequence as a temporal chain, where its states are tip locations; given candidate tip locations, actin filaments' body points are inferred by a dynamic programming method, which adaptively generates candidate solutions. Combining candidate tip locations and their inferred body points, the temporal chain model is efficiently optimized using another dynamic programming method. Evaluation on noisy TIRFM image sequences demonstrates the accuracy and robustness of this approach. PMID:21761674

  13. Spontaneous actin dynamics in contractile rings

    NASA Astrophysics Data System (ADS)

    Kruse, Karsten; Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Riveline, Daniel

    Networks of polymerizing actin filaments are known to be capable to self-organize into a variety of structures. For example, spontaneous actin polymerization waves have been observed in living cells in a number of circumstances, notably, in crawling neutrophils and slime molds. During later stages of cell division, they can also spontaneously form a contractile ring that will eventually cleave the cell into two daughter cells. We present a framework for describing networks of polymerizing actin filaments, where assembly is regulated by various proteins. It can also include the effects of molecular motors. We show that the molecular processes driven by these proteins can generate various structures that have been observed in contractile rings of fission yeast and mammalian cells. We discuss a possible functional role of each of these patterns. The work was supported by Agence Nationale de la Recherche, France, (ANR-10-LABX-0030-INRT) and by Deutsche Forschungsgemeinschaft through SFB1027.

  14. Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

    PubMed Central

    Hoffmann, Céline; Moreau, Flora; Moes, Michèle; Luthold, Carole; Dieterle, Monika; Goretti, Emeline; Neumann, Katrin; Steinmetz, André

    2014-01-01

    The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking. PMID:24934443

  15. Fatal congenital myopathy with actin filament deposits.

    PubMed

    Bornemann, A; Petersen, M B; Schmalbruch, H

    1996-07-01

    We present the clinical and morphological findings in a case of progressive congenital myopathy. The symptoms present at birth included severe general muscular hypotonia, diffuse muscular atrophy, arthrogryposis, absence of spontaneous movements, and left ventricular hypertrophy. A biopsy specimen taken from the gastrocnemius muscle when the patient was 2 weeks old revealed deposits which consisted of actin filaments as shown by electron microscopy. The infant was occasionally respirator dependent but was mostly able to breathe unassisted. At the age of 5 months he died of respiratory failure. The actin filament deposits may explain the clinical findings.

  16. Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

    PubMed Central

    Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.

    2015-01-01

    Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073

  17. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

    Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

    2011-09-01

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins. PMID:21757693

  18. Actin nucleators in the nucleus: an emerging theme.

    PubMed

    Weston, Louise; Coutts, Amanda S; La Thangue, Nicholas B

    2012-08-01

    Actin is an integral component of the cytoskeleton, forming a plethora of macromolecular structures that mediate various cellular functions. The formation of such structures relies on the ability of actin monomers to associate into polymers, and this process is regulated by actin nucleation factors. These factors use monomeric actin pools at specific cellular locations, thereby permitting rapid actin filament formation when required. It has now been established that actin is also present in the nucleus, where it is implicated in chromatin remodelling and the regulation of eukaryotic gene transcription. Notably, the presence of typical actin filaments in the nucleus has not been demonstrated directly. However, studies in recent years have provided evidence for the nuclear localisation of actin nucleation factors that promote cytoplasmic actin polymerisation. Their localisation to the nucleus suggests that these proteins mediate collaboration between the cytoskeleton and the nucleus, which might be dependent on their ability to promote actin polymerisation. The nature of this cooperation remains enigmatic and it will be important to elucidate the physiological relevance of the link between cytoskeletal actin networks and nuclear events. This Commentary explores the current evidence for the nuclear roles of actin nucleation factors. Furthermore, the implication of actin-associated proteins in relaying exogenous signals to the nucleus, particularly in response to cellular stress, will be considered.

  19. Dynamic Regulation of Sarcomeric Actin Filaments in Striated Muscle

    PubMed Central

    Ono, Shoichiro

    2010-01-01

    In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin subunits within sarcomeric actin filaments are dynamically exchanged without altering overall sarcomeric structures. A number of regulators for actin dynamics have been identified, and malfunction of these regulators often result in disorganization of myofibril structures or muscle diseases. Therefore, proper regulation of actin dynamics in striated muscle is critical for assembly and maintenance of functional myofibrils. Recent studies have suggested that both enhancers of actin dynamics and stabilizers of actin filaments are important for sarcomeric actin organization. Further investigation of the regulatory mechanism of actin dynamics in striated muscle should be a key to understanding how myofibrils develop and operate. © 2010 Wiley-Liss, Inc. PMID:20737540

  20. Actin3 promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes.

    PubMed

    Jásik, Ján; Mičieta, Karol; Siao, Wei; Voigt, Boris; Stuchlík, Stanislav; Schmelzer, Elmon; Turňa, Ján; Baluška, František

    2016-01-01

    The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific Actin3 promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific Actin3 promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical 'actin collars' or 'fringes' are absent.

  1. Actin3 promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes

    PubMed Central

    Jásik, Ján; Mičieta, Karol; Siao, Wei; Voigt, Boris; Stuchlík, Stanislav; Schmelzer, Elmon; Turňa, Ján; Baluška, František

    2016-01-01

    ABSTRACT The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific Actin3 promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific Actin3 promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical ‘actin collars’ or ‘fringes’ are absent. PMID:26980067

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

    PubMed

    Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

    1998-06-01

    Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin

  3. Influence of thin porous Al{sub 2}O{sub 3} layer on aluminum cathode to the H{sub a}lpha line shape in glow discharge

    SciTech Connect

    Steflekova, V.; Sisovic, N. M.; Konjevic, N.

    2009-06-01

    The results of the Balmer alfa line shape study in a plane cathode-hollow anode Grimm discharge with aluminum (Al) cathode covered with thin layer of porous Al{sub 2}O{sub 3} are presented. The comparison with same line profile recorded with pure Al cathode shows lack of excessive Doppler broadened line wings, which are always detected in glow discharge with metal cathode. The effect is explained by the lack of strong electric field in the cathode sheath region, which is missing in the presence of thin oxide layer in, so called, spray discharge.

  4. The actin cytoskeleton as a sensor and mediator of apoptosis

    PubMed Central

    Desouza, Melissa; Gunning, Peter W.; Stehn, Justine R.

    2012-01-01

    Apoptosis is an important biological process required for the removal of unwanted or damaged cells. Mounting evidence implicates the actin cytoskeleton as both a sensor and mediator of apoptosis. Studies also suggest that actin binding proteins (ABPs) significantly contribute to apoptosis and that actin dynamics play a key role in regulating apoptosis signaling. Changes in the organization of the actin cytoskeleton has been attributed to the process of malignant transformation and it is hypothesized that remodeling of the actin cytoskeleton may enable tumor cells to evade normal apoptotic signaling. This review aims to illuminate the role of the actin cytoskeleton in apoptosis by systematically analyzing how actin and ABPs regulate different apoptosis pathways and to also highlight the potential for developing novel compounds that target tumor-specific actin filaments. PMID:22880146

  5. CNS myelin wrapping is driven by actin disassembly.

    PubMed

    Zuchero, J Bradley; Fu, Meng-Meng; Sloan, Steven A; Ibrahim, Adiljan; Olson, Andrew; Zaremba, Anita; Dugas, Jason C; Wienbar, Sophia; Caprariello, Andrew V; Kantor, Christopher; Leonoudakis, Dmitri; Leonoudakus, Dmitri; Lariosa-Willingham, Karen; Kronenberg, Golo; Gertz, Karen; Soderling, Scott H; Miller, Robert H; Barres, Ben A

    2015-07-27

    Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins and rapid disassembly of the oligodendrocyte actin cytoskeleton and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together, these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility.

  6. Actin network architecture can determine myosin motor activity.

    PubMed

    Reymann, Anne-Cécile; Boujemaa-Paterski, Rajaa; Martiel, Jean-Louis; Guérin, Christophe; Cao, Wenxiang; Chin, Harvey F; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2012-06-01

    The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

  7. Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin

    PubMed Central

    Huang, Shuxian; Umemoto, Ryo; Tamura, Yuki; Kofuku, Yutaka; Uyeda, Taro Q. P.; Nishida, Noritaka; Shimada, Ichio

    2016-01-01

    Actin cytoskeleton dynamics are controlled by various actin binding proteins (ABPs) that modulate the polymerization of the monomeric G-actin and the depolymerization of filamentous F-actin. Although revealing the structures of the actin/ABP complexes is crucial to understand how the ABPs regulate actin dynamics, the X-ray crystallography and cryoEM methods are inadequate to apply for the ABPs that interact with G- or F-actin with lower affinity or multiple binding modes. In this study, we aimed to establish the alternative method to build a structural model of G-actin/ABP complexes, utilizing the paramagnetic relaxation enhancement (PRE) experiments. Thymosin β4 (Tβ4) was used as a test case for validation, since its structure in complex with G-actin was reported recently. Recombinantly expressed G-actin, containing a cysteine mutation, was conjugated with a nitroxyl spin label at the specific site. Based on the intensity ratio of the 1H-15N HSQC spectra of Tβ4 in the complex with G-actin in the paramagnetic and diamagnetic states, the distances between the amide groups of Tβ4 and the spin label of G-actin were estimated. Using the PRE-derived distance constraints, we were able to compute a well-converged docking structure of the G-actin/Tβ4 complex that shows great accordance with the reference structure. PMID:27654858

  8. Performance of actinic EUVL mask imaging using a zoneplate microscope

    SciTech Connect

    Goldberg, K; Naulleau, P; Barty, A; Rekawa, S; Kemp, C; Gunion, R; Salmassi, F; Gullikson, E; Anderson, E; Han, H

    2007-09-25

    The SEMATECH Berkeley Actinic Inspection Tool (AIT) is a dual-mode, scanning and imaging extreme-ultraviolet (EUV) microscope designed for pre-commercial EUV mask research. Dramatic improvements in image quality have been made by the replacement of several critical optical elements, and the introduction of scanning illumination to improve uniformity and contrast. We report high quality actinic EUV mask imaging with resolutions as low as 100-nm half-pitch, (20-nm, 5x wafer equivalent size), and an assessment of the imaging performance based on several metrics. Modulation transfer function (MTF) measurements show high contrast imaging for features sizes close to the diffraction-limit. An investigation of the illumination coherence shows that AIT imaging is much more coherent than previously anticipated, with {sigma} below 0.2. Flare measurements with several line-widths show a flare contribution on the order of 2-3% relative intensity in dark regions above the 1.3% absorber reflectivity on the test mask used for these experiments. Astigmatism coupled with focal plane tilt are the dominant aberrations we have observed. The AIT routinely records 250-350 high-quality images in numerous through-focus series per 8-hour shift. Typical exposure times range from 0.5 seconds during alignment, to approximately 20 seconds for high-resolution images.

  9. Performance of actinic EUVL mask imaging using a zoneplatemicroscope

    SciTech Connect

    Goldberg, Kenneth A.; Naulleau, Patrick P.; Barty, Anton; Rekawa,Senajith B.; Kemp, Charles D.; Gunion, Robert F.; Salmassi, Farhad; Gullikson, Eric M.; Anderson, Erik H.; Han, Hak-Seung

    2007-08-20

    The SEMATECH Berkeley Actinic Inspection Tool (AIT) is a dual-mode, scanning and imaging extreme-ultraviolet (EUV) microscope designed for pre-commercial EUV mask research. Dramatic improvements in image quality have been made by the replacement of several critical optical elements, and the introduction of scanning illumination to improve uniformity and contrast. We report high quality actinic EUV mask imaging with resolutions as low as 100-nm half-pitch, (20-nm, 5x wafer equivalent size), and an assessment of the imaging performance based on several metrics. Modulation transfer function (MTF) measurements show high contrast imaging for features sizes close to the diffraction-limit. An investigation of the illumination coherence shows that AIT imaging is much more coherent than previously anticipated, with {sigma} below 0.2. Flare measurements with several line-widths show a flare contribution on the order of 2-3% relative intensity in dark regions above the 1.3% absorber reflectivity on the test mask used for these experiments. Astigmatism coupled with focal plane tilt are the dominant aberrations we have observed. The AIT routinely records 250-350 high-quality images in numerous through-focus series per 8-hour shift. Typical exposure times range from 0.5 seconds during alignment, to approximately 20 seconds for high-resolution images.

  10. Interaction of profilin with the barbed end of actin filaments.

    PubMed

    Courtemanche, Naomi; Pollard, Thomas D

    2013-09-17

    Profilin binds not only to actin monomers but also to the barbed end of the actin filament, where it inhibits association of subunits. To address open questions about the interactions of profilin with barbed ends, we measured the effects of a wide range of concentrations of Homo sapiens profilin 1 on the rate of elongation of individual skeletal muscle actin filaments by total internal reflection fluorescence microscopy. Much higher concentrations of profilin were required to stop elongation by AMP-PNP-actin monomers than ADP-actin monomers. High concentrations of profilin depolymerized barbed ends at a rate much faster than the spontaneous dissociation rates of Mg-ATP-, Mg-AMP-PNP-, Mg-ADP-Pi-, and Mg-ADP-actin subunits. Fitting a thermodynamic model to these data allowed us to determine the affinities of profilin and profilin-actin for barbed ends and the influence of the nucleotide bound to actin on these interactions. Profilin has a much higher affinity for ADP-actin filament barbed ends (Kd = 1 μM) than AMP-PNP-actin filament barbed ends (Kd = 226 μM). ADP-actin monomers associated with profilin bind to ADP-actin filament barbed ends 10% as fast as free ADP-actin monomers, but bound profilin does not affect the rate of association of AMP-PNP-actin monomers with barbed ends. The differences in the affinities of AMP-PNP- and ADP-bound barbed ends for profilin and profilin-actin suggest that conformations of barbed end subunits differ from those of monomers and change upon nucleotide hydrolysis and phosphate release. A structural model revealed minor steric clashes between profilin and actin subunits at the barbed end that explain the biochemical results.

  11. Actin of Beta vulgaris seedlings under the clinorotation

    NASA Astrophysics Data System (ADS)

    Kozeko, L. Ye.

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

  12. Curvature and torsion in growing actin networks

    NASA Astrophysics Data System (ADS)

    Shaevitz, Joshua W.; Fletcher, Daniel A.

    2008-06-01

    Intracellular pathogens such as Listeria monocytogenes and Rickettsia rickettsii move within a host cell by polymerizing a comet-tail of actin fibers that ultimately pushes the cell forward. This dense network of cross-linked actin polymers typically exhibits a striking curvature that causes bacteria to move in gently looping paths. Theoretically, tail curvature has been linked to details of motility by considering force and torque balances from a finite number of polymerizing filaments. Here we track beads coated with a prokaryotic activator of actin polymerization in three dimensions to directly quantify the curvature and torsion of bead motility paths. We find that bead paths are more likely to have low rather than high curvature at any given time. Furthermore, path curvature changes very slowly in time, with an autocorrelation decay time of 200 s. Paths with a small radius of curvature, therefore, remain so for an extended period resulting in loops when confined to two dimensions. When allowed to explore a three-dimensional (3D) space, path loops are less evident. Finally, we quantify the torsion in the bead paths and show that beads do not exhibit a significant left- or right-handed bias to their motion in 3D. These results suggest that paths of actin-propelled objects may be attributed to slow changes in curvature, possibly associated with filament debranching, rather than a fixed torque.

  13. Viscoelastic properties of actin-coated membranes

    NASA Astrophysics Data System (ADS)

    Helfer, E.; Harlepp, S.; Bourdieu, L.; Robert, J.; Mackintosh, F. C.; Chatenay, D.

    2001-02-01

    In living cells, cytoskeletal filaments interact with the plasma membrane to form structures that play a key role in cell shape and mechanical properties. To study the interaction between these basic components, we designed an in vitro self-assembled network of actin filaments attached to the outer surface of giant unilamellar vesicles. Optical tweezers and single-particle tracking experiments are used to study the rich dynamics of these actin-coated membranes (ACM). We show that microrheology studies can be carried out on such an individual microscopic object. The principle of the experiment consists in measuring the thermally excited position fluctuations of a probe bead attached biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin network modifies strongly the membrane dynamics with respect to a fluid, lipid bilayer one. It induces first a finite (ω=0) two-dimensional (2D) shear modulus G02D~0.5 to 5 μN/m in the membrane plane. Moreover, the frequency dependence at high frequency of the shear modulus [G'2D(f )~f0.85+/-0.07] and of the bending modulus (κACM(f)~f0.55+/-0.21) demonstrate the viscoelastic behavior of the composite membrane. These results are consistent with a common exponent of 0.75 for both moduli as expected from our model and from prior measurements on actin solutions.

  14. Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

    PubMed

    Schnoor, Michael

    2015-04-15

    The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

  15. Competition of two distinct actin networks for actin defines a bistable switch for cell polarization

    PubMed Central

    Lomakin, Alexis J.; Lee, Kun-Chun; Han, Sangyoon J.; Bui, D A.; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-01-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype upon relaxation of the actomyosin cytoskeleton. We find that myosin-II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. At low contractility regimes epithelial cells polarize in a front-back manner due to emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin-II from the front to the back of the cell, where the motor locally “locks” actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high contractility-driven cell motion is inefficient. PMID:26414403

  16. Actin-organising properties of the muscular dystrophy protein myotilin.

    PubMed

    von Nandelstadh, Pernilla; Grönholm, Mikaela; Moza, Monica; Lamberg, Arja; Savilahti, Harri; Carpén, Olli

    2005-10-15

    Myotilin is a sarcomeric Z-disc protein that binds F-actin directly and bundles actin filaments, although it does not contain a conventional actin-binding domain. Expression of mutant myotilin leads to sarcomeric alterations in the dominantly inherited limb-girdle muscular dystrophy 1A and in myofibrillar myopathy/desmin-related myopathy. Together, with previous in vitro studies, this indicates that myotilin has an important function in the assembly and maintenance of Z-discs. This study characterises further the interaction between myotilin and actin. Functionally important regions in myotilin were identified by actin pull-down and yeast two-hybrid assays and with a novel strategy that combines in vitro DNA transposition-based peptide insertion mutagenesis with phenotype analysis in yeast cells. The shortest fragment to bind actin was the second Ig domain together with a short C-terminal sequence. Concerted action of the first and second Ig domain was, however, necessary for the functional activity of myotilin, as verified by analysis of transposon mutants, actin binding and phenotypic effect in mammalian cells. Furthermore, the Ig domains flanked with N- and C-terminal regions were needed for actin-bundling, indicating that the mere actin-binding sequence was insufficient for the actin-regulating activity. None of the four known disease-associated mutations altered the actin-organising ability. These results, together with previous studies in titin and kettin, identify the Ig domain as an actin-binding unit.

  17. Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis.

    PubMed

    Spracklen, Andrew J; Kelpsch, Daniel J; Chen, Xiang; Spracklen, Cassandra N; Tootle, Tina L

    2014-02-01

    Prostaglandins (PGs)--lipid signals produced downstream of cyclooxygenase (COX) enzymes--regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton--temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling.

  18. Shape control of lipid bilayer membranes by confined actin bundles.

    PubMed

    Tsai, Feng-Ching; Koenderink, Gijsje Hendrika

    2015-12-01

    In living cells, lipid membranes and biopolymers determine each other's conformation in a delicate force balance. Cellular polymers such as actin filaments are strongly confined by the plasma membrane in cell protrusions such as lamellipodia and filopodia. Conversely, protrusion formation is facilitated by actin-driven membrane deformation and these protrusions are maintained by dense actin networks or bundles of actin filaments. Here we investigate the mechanical interplay between actin bundles and lipid bilayer membranes by reconstituting a minimal model system based on cell-sized liposomes with encapsulated actin filaments bundled by fascin. To address the competition between the deformability of the membrane and the enclosed actin bundles, we tune the bundle stiffness (through the fascin-to-actin molar ratio) and the membrane rigidity (through protein decoration). Using confocal microscopy and quantitative image analysis, we show that actin bundles deform the liposomes into a rich set of morphologies. For liposomes having a small membrane bending rigidity, the actin bundles tend to generate finger-like membrane protrusions that resemble cellular filopodia. Stiffer bundles formed at high crosslink density stay straight in the liposome body, whereas softer bundles formed at low crosslink density are bent and kinked. When the membrane has a large bending rigidity, membrane protrusions are suppressed. In this case, membrane enclosure forces the actin bundles to organize into cortical rings, to minimize the energy cost associated with filament bending. Our results highlight the importance of taking into account mechanical interactions between the actin cytoskeleton and the membrane to understand cell shape control.

  19. Unconventional actins and actin-binding proteins in human protozoan parasites.

    PubMed

    Gupta, C M; Thiyagarajan, S; Sahasrabuddhe, A A

    2015-06-01

    Actin and its regulatory proteins play a key role in several essential cellular processes such as cell movement, intracellular trafficking and cytokinesis in most eukaryotes. While these proteins are highly conserved in higher eukaryotes, a number of unicellular eukaryotic organisms contain divergent forms of these proteins which have highly unusual biochemical and structural properties. Here, we review the biochemical and structural properties of these unconventional actins and their core binding proteins which are present in commonly occurring human protozoan parasites.

  20. FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongation

    SciTech Connect

    Thompson, Morgan E; Heimsath, Ernest G; Gauvin, Timothy J; Higgs, Henry N; Kull, F Jon

    2012-12-09

    Formins are actin-assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation through interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4-Å structure of a mouse FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2 actin-binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding, whereas those in the lasso-post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2-mediated filament elongation by processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus.

  1. Application of the spine-layer jet radiation model to outbursts in the broad-line radio galaxy 3C 120

    NASA Astrophysics Data System (ADS)

    Janiak, M.; Sikora, M.; Moderski, R.

    2016-05-01

    We present a detailed Fermi/LAT data analysis for the broad-line radio galaxy 3C 120. This source has recently entered into a state of increased γ-ray activity which manifested itself in two major flares detected by Fermi/LAT in 2014 September and 2015 April with no significant flux changes reported in other wavelengths. We analyse available data focusing our attention on aforementioned outbursts. We find very fast variability time-scale during flares (of the order of hours) together with a significant γ-ray flux increase. We show that the ˜6.8 yr averaged γ-ray emission of 3C 120 is likely a sum of the external radiation Compton and the synchrotron self-Compton radiative components. To address the problem of violent γ-ray flares and fast variability we model the jet radiation dividing the jet structure into two components: the wide and relatively slow outer layer and the fast, narrow spine. We show that with the addition of the fast spine occasionally bent towards the observer we are able to explain observed spectral energy distribution of 3C 120 during flares with the Compton upscattered broad-line region and dusty torus photons as main γ-rays emission mechanism.

  2. Biomechanics of Schlemm's canal endothelial cells: influence on F-actin architecture.

    PubMed

    Ethier, C Ross; Read, A Thomas; Chan, Darren

    2004-10-01

    Aqueous humor drains from the eye through Schlemm's canal, a small endothelial-lined collecting duct. Schlemm's canal endothelial cells may be important in controlling the pressure within the eye (and hence are of interest in glaucoma), and are subject to an unusual combination of shear stress and a basal-to-apical pressure gradient. We sought to characterize this biomechanical environment and determine its effects on F-actin architecture in situ. A theoretical model of flow in Schlemm's canal was used to estimate shear stresses applied to endothelial cells by flowing aqueous humor. Alignment of Schlemm's canal endothelial cells in human eyes was quantified by scanning electron microscopy. F-actin architecture was visualized by fluorescent labeling and compared for closely adjacent cells exposed to different biomechanical environments. We found that, despite the relatively low flow rate of aqueous humor, shear stresses experienced by Schlemm's canal endothelial cells could reach those in the arterial system. Schlemm's canal endothelial cells showed a statistically significant preferential alignment, consistent with a shear-mediated effect. Schlemm's canal endothelial cells subjected to a basal-to-apical pressure gradient due to transendothelial flow showed a prominent marginal band of F-actin with relatively few cytoplasmic filaments. Adjacent cells not subject to this gradient showed little marginal F-actin, with a denser cytoplasmic random network. We conclude that Schlemm's canal endothelial cells experience physiologically significant levels of shear stress, promoting cell alignment. We speculate that this may help control the calibre of Schlemm's canal. F-actin distribution depends critically on the presence or absence of transendothelial flow and its associated pressure gradient. In the case of this pressure gradient, mechanical reinforcement around the cell periphery by F-actin seems to be critical. PMID:15454474

  3. Structure of a Bud6/actin complex reveals a novel WH2-like actin monomer recruitment motif

    PubMed Central

    Park, Eunyoung; Graziano, Brian R.; Zheng, Wei; Garabedian, Mikael; Goode, Bruce L.; Eck, Michael J.

    2015-01-01

    SUMMARY In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a “core” domain that binds to the formin and a “flank” domain that binds monomeric actin. Here we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6flank interact with actin; one binds in a groove at the barbed-end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation. PMID:26118535

  4. Structure of a Bud6/Actin Complex Reveals a Novel WH2-like Actin Monomer Recruitment Motif.

    PubMed

    Park, Eunyoung; Graziano, Brian R; Zheng, Wei; Garabedian, Mikael; Goode, Bruce L; Eck, Michael J

    2015-08-01

    In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a "core" domain that binds to the formin and a "flank" domain that binds monomeric actin. Here, we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6(flank) interact with actin; one binds in a groove at the barbed end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation.

  5. The origin and evolution of green algal and plant actins.

    PubMed

    An, S S; Möpps, B; Weber, K; Bhattacharya, D

    1999-02-01

    The Viridiplantae are subdivided into two groups: the Chlorophyta, which includes the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Prasinophyceae; and the Streptophyta, which includes the Charophyceae and all land plants. Within the Streptophyta, the actin genes of the angiosperms diverge nearly simultaneously from each other before the separation of monocots and dicots. Previous evolutionary analyses have provided limited insights into the gene duplications that have produced these complex gene families. We address the origin and diversification of land plant actin genes by studying the phylogeny of actins within the green algae, ferns, and fern allies. Partial genomic sequences or cDNAs encoding actin were characterized from Cosmarium botrytis (Zygnematales), Selaginella apoda (Selaginellales), Anemia phyllitidis (Polypodiales), and Psilotum triquetrum (Psilotales). Selaginella contains at least two actin genes. One sequence (Ac2) diverges within a group of fern sequences that also includes the Psilotum Ac1 actin gene and one gymnosperm sequence (Cycas revoluta Cyc3). This clade is positioned outside of the angiosperm actin gene radiation. The second Selaginella sequence (Ac1) is the sister to all remaining land plant actin sequences, although the internal branches in this portion of the tree are very short. Use of complete actin-coding regions in phylogenetic analyses provides support for the separation of angiosperm actins into two classes. N-terminal "signature" sequence analyses support these groupings. One class (VEG) includes actin genes that are often expressed in vegetative structures. The second class (REP) includes actin genes that trace their ancestry within the vegetative actins and contains members that are largely expressed in reproductive structures. Analysis of intron positions within actin genes shows that sequences from both Selaginella and Cosmarium contain the conserved 20-3, 152-1, and 356-3 introns found in many members of the

  6. Demonstration of prominent actin filaments in the root columella.

    PubMed

    Collings, D A; Zsuppan, G; Allen, N S; Blancaflor, E B

    2001-02-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling. PMID:11289604

  7. Exploring the Stability Limits of Actin and Its Suprastructures

    PubMed Central

    Rosin, Christopher; Erlkamp, Mirko; Ecken, Julian von der; Raunser, Stefan; Winter, Roland

    2014-01-01

    Actin is the main component of the microfilament system in eukaryotic cells and can be found in distinct morphological states. Global (G)-actin is able to assemble into highly organized, supramolecular cellular structures known as filamentous (F)-actin and bundled (B)-actin. To evaluate the structure and stability of G-, F-, and B-actin over a wide range of temperatures and pressures, we used Fourier transform infrared spectroscopy in combination with differential scanning and pressure perturbation calorimetry, small-angle x-ray scattering, laser confocal scanning microscopy, and transmission electron microscopy. Our analysis was designed to provide new (to our knowledge) insights into the stabilizing forces of actin self-assembly and to reveal the stability of the actin polymorphs, including in conditions encountered in extreme environments. In addition, we sought to explain the limited pressure stability of actin self-assembly observed in vivo. G-actin is not only the least temperature-stable but also the least pressure-stable actin species. Under abyssal conditions, where temperatures as low as 1–4°C and pressures up to 1 kbar are reached, G-actin is hardly stable. However, the supramolecular assemblies of actin are stable enough to withstand the extreme conditions usually encountered on Earth. Beyond ∼3–4 kbar, filamentous structures disassemble, and beyond ∼4 kbar, complete dissociation of F-actin structures is observed. Between ∼1 and 2 kbar, some disordering of actin assemblies commences, in agreement with in vivo observations. The limited pressure stability of the monomeric building block seems to be responsible for the suppression of actin assembly in the kbar pressure range. PMID:25517163

  8. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  9. Arabidopsis VILLIN2 and VILLIN3 are required for the generation of thick actin filament bundles and for directional organ growth.

    PubMed

    van der Honing, Hannie S; Kieft, Henk; Emons, Anne Mie C; Ketelaar, Tijs

    2012-03-01

    In plant cells, actin filament bundles serve as tracks for myosin-dependent organelle movement and play a role in the organization of the cytoplasm. Although virtually all plant cells contain actin filament bundles, the role of the different actin-bundling proteins remains largely unknown. In this study, we investigated the role of the actin-bundling protein villin in Arabidopsis (Arabidopsis thaliana). We used Arabidopsis T-DNA insertion lines to generate a double mutant in which VILLIN2 (VLN2) and VLN3 transcripts are truncated. Leaves, stems, siliques, and roots of vln2 vln3 double mutant plants are twisted, which is caused by local differences in cell length. Microscopy analysis of the actin cytoskeleton showed that in these double mutant plants, thin actin filament bundles are more abundant while thick actin filament bundles are virtually absent. In contrast to full-length VLN3, truncated VLN3 lacking the headpiece region does not rescue the phenotype of the vln2 vln3 double mutant. Our results show that villin is involved in the generation of thick actin filament bundles in several cell types and suggest that these bundles are involved in the regulation of coordinated cell expansion.

  10. Symbiont-Induced Changes in Host Actin during the Onset of a Beneficial Animal-Bacterial Association

    PubMed Central

    Kimbell, Jennifer R.; McFall-Ngai, Margaret J.

    2004-01-01

    The influence of bacteria on the cytoskeleton of animal cells has been studied extensively only in pathogenic associations. We characterized changes in host cytoskeletal actin induced by the bacterial partner during the onset of a cooperative animal-bacteria association using the squid-vibrio model. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that Vibrio fischeri induced a dramatic increase in actin protein abundance in the bacteria-associated host tissues during the onset of the symbiosis. Immunocytochemistry revealed that this change in actin abundance correlated with a two- to threefold increase in actin in the apical cell surface of the epithelium-lined ducts, the route of entry of symbionts into host tissues. Real-time reverse transcriptase PCR and in situ hybridization did not detect corresponding changes in actin mRNA. Temporally correlated with the bacteria-induced changes in actin levels was a two- to threefold decrease in duct circumference, a 20% loss in the average number of cells interfacing with the duct lumina, and dramatic changes in duct cell shape. When considered with previous studies of the biomechanical and biochemical characteristics of the duct, these findings suggest that the bacterial symbionts, upon colonizing the host organ, induce modifications that physically and chemically limit the opportunity for subsequent colonizers to pass through the ducts. Continued study of the squid-vibrio system will allow further comparisons of the mechanisms by which pathogenic and cooperative bacteria influence cytoskeleton dynamics in host cells. PMID:15006763

  11. Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton.

    PubMed

    Dombroski, Derek; Houghtling, Richard A; Labno, Christine M; Precht, Patricia; Takesono, Aya; Caplen, Natasha J; Billadeau, Daniel D; Wange, Ronald L; Burkhardt, Janis K; Schwartzberg, Pamela L

    2005-02-01

    The Tec family kinase Itk is an important regulator of Ca(2+) mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.

  12. Actin nucleation and elongation factors: mechanisms and interplay.

    PubMed

    Chesarone, Melissa A; Goode, Bruce L

    2009-02-01

    Cells require actin nucleators to catalyze the de novo assembly of filaments and actin elongation factors to control the rate and extent of polymerization. Nucleation and elongation factors identified to date include Arp2/3 complex, formins, Ena/VASP, and newcomers Spire, Cobl, and Lmod. Here, we discuss recent advances in understanding their activities and mechanisms and new evidence for their cooperation and interaction in vivo. Earlier models had suggested that different nucleators function independently to assemble distinct actin arrays. However, more recent observations indicate that the construction of most cellular actin networks depends on the activities of multiple actin assembly-promoting factors working in concert.

  13. Direct Observation of Tropomyosin Binding to Actin Filaments

    PubMed Central

    Schmidt, William M.; Lehman, William; Moore, Jeffrey R.

    2015-01-01

    Tropomyosin is an elongated α-helical coiled-coil that binds to seven consecutive actin subunits along the long-pitch helix of actin filaments. Once bound, tropomyosin polymerizes end-to-end and both stabilizes F-actin and regulates access of various actin binding proteins including myosin in muscle and non-muscle cells. Single tropomyosin molecules bind weakly to F-actin with millimolar Kd, whereas the end-to-end linked tropomyosin associates with about a one thousand-fold greater affinity. Despite years of study, the assembly mechanism of tropomyosin onto actin filaments remains unclear. In the current study, we used total internal reflection fluorescence (TIRF) microscopy to directly monitor the cooperative binding of fluorescently labeled tropomyosin molecules to phalloidin-stabilized actin filaments. We find that tropomyosin molecules assemble from multiple growth sites following random low affinity binding of single molecules to actin. As the length of the tropomyosin chain increases, the probability of detachment decreases, which leads to further chain growth. Tropomyosin chain extension is linearly dependent on tropomyosin concentration, occurring at approximately 100 monomers/(μM*s). The random tropomyosin binding to F-actin leads to discontinuous end-to-end association where gaps in the chain continuity smaller than the required seven sequential actin monomers are available. Direct observation of tropomyosin detachment revealed the number of gaps in actin-bound tropomyosin, the time course of gap annealing, and the eventual filament saturation process. PMID:26033920

  14. Actin in xenopus oocytes: II. intracellular distribution and polymerizability

    PubMed Central

    Merriam, RW; Clark, TG

    1978-01-01

    The largest oocytes of Xenopus Laevis were broken open in the absence of shearing forces which might transfer actin from particulate to supernatant fractions. Particulate and postmitochondrial supernatant fractions were prepared by centrifugation. SDS-electrophoretic fractionation on polyacrylamide gels and quantitative scanning techniques were used to separate actin and to assay its amount in cellular fractions. The actin has been identified in electrophoretograms by its molecular weight and its binding to DNase I. oocytes contain 1.4-1.7 {um}g of actin per cell, of which up to 88 percent is recovered in the postmitochondrial supernate under a variety of conditions. In the soluble fraction, it represents about 8.8 percent of the total protein. Its concentration in native cytoplasm was directly assayed at 4.1 mg/ml. There is no detectable actin that can be transferred from the particulate to the soluble phase by neutral detergents or ionic conditions that would depolymerize muscle actin. Centrifugation of the soluble oocyte fractions showed that 75-95 percent of the actin can not be sedimented under forces that would pellet filamentous actin. Addition of potassium and magnesium to the cytoplasm, to concentrations that would polymerize muscle actin, does not increase the amount of sedimentable actin. Roughly one-third of the soluble actin is recovered from Sephadex columns at about the position of monomer. About two- thirds is in complexes of 100,000 daltons or greater. PMID:565782

  15. Distributed actin turnover in the lamellipodium and FRAP kinetics.

    PubMed

    Smith, Matthew B; Kiuchi, Tai; Watanabe, Naoki; Vavylonis, Dimitrios

    2013-01-01

    Studies of actin dynamics at the leading edge of motile cells with single-molecule speckle (SiMS) microscopy have shown a broad distribution of EGFP-actin speckle lifetimes and indicated actin polymerization and depolymerization over an extended region. Other experiments using FRAP with the same EGFP-actin as a probe have suggested, by contrast, that polymerization occurs exclusively at the leading edge. We performed FRAP experiments on XTC cells to compare SiMS to FRAP on the same cell type. We used speckle statistics obtained by SiMS to model the steady-state distribution and kinetics of actin in the lamellipodium. We demonstrate that a model with a single diffuse actin species is in good agreement with FRAP experiments. A model including two species of diffuse actin provides an even better agreement. The second species consists of slowly diffusing oligomers that associate to the F-actin network throughout the lamellipodium or break up into monomers after a characteristic time. Our work motivates studies to test the presence and composition of slowly diffusing actin species that may contribute to local remodeling of the actin network and increase the amount of soluble actin.

  16. Bundling actin filaments from membranes: some novel players

    PubMed Central

    Thomas, Clément

    2012-01-01

    Progress in live-cell imaging of the cytoskeleton has significantly extended our knowledge about the organization and dynamics of actin filaments near the plasma membrane of plant cells. Noticeably, two populations of filamentous structures can be distinguished. On the one hand, fine actin filaments which exhibit an extremely dynamic behavior basically characterized by fast polymerization and prolific severing events, a process referred to as actin stochastic dynamics. On the other hand, thick actin bundles which are composed of several filaments and which are comparatively more stable although they constantly remodel as well. There is evidence that the actin cytoskeleton plays critical roles in trafficking and signaling at both the cell cortex and organelle periphery but the exact contribution of actin bundles remains unclear. A common view is that actin bundles provide the long-distance tracks used by myosin motors to deliver their cargo to growing regions and accordingly play a particularly important role in cell polarization. However, several studies support that actin bundles are more than simple passive highways and display multiple and dynamic roles in the regulation of many processes, such as cell elongation, polar auxin transport, stomatal and chloroplast movement, and defense against pathogens. The list of identified plant actin-bundling proteins is ever expanding, supporting that plant cells shape structurally and functionally different actin bundles. Here I review the most recently characterized actin-bundling proteins, with a particular focus on those potentially relevant to membrane trafficking and/or signaling. PMID:22936939

  17. A second Las17 monomeric actin-binding motif functions in Arp2/3-dependent actin polymerization during endocytosis.

    PubMed

    Feliciano, Daniel; Tolsma, Thomas O; Farrell, Kristen B; Aradi, Al; Di Pietro, Santiago M

    2015-04-01

    During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.

  18. The Potential Roles of Actin in The Nucleus

    PubMed Central

    Falahzadeh, Khadijeh; Banaei-Esfahani, Amir; Shahhoseini, Maryam

    2015-01-01

    Over the past few decades, actin’s presence in the nucleus has been demonstrated. Actin is a key protein necessary for different nuclear processes. Although actin is well known for its functional role in dynamic behavior of the cytoskeleton, emerging studies are now highlighting new roles for actin. At the present time there is no doubt about the presence of actin in the nucleus. A number of studies have uncovered the functional involvement of actin in nuclear processes. Actin as one of the nuclear components has its own structured and functional rules, such as nuclear matrix association, chromatin remodeling, transcription by RNA polymerases I, II, III and mRNA processing. In this historical review, we attempt to provide an overview of our current understanding of the functions of actin in the nucleus. PMID:25870830

  19. Regulation of cellular actin architecture by S100A10.

    PubMed

    Jung, M Juliane; Murzik, Ulrike; Wehder, Liane; Hemmerich, Peter; Melle, Christian

    2010-04-15

    Actin structures are involved in several biological processes and the disruption of actin polymerisation induces impaired motility of eukaryotic cells. Different factors are involved in regulation and maintenance of the cytoskeletal actin architecture. Here we show that S100A10 participates in the particular organisation of actin filaments. Down-regulation of S100A10 by specific siRNA triggered a disorganisation of filamentous actin structures without a reduction of the total cellular actin concentration. In contrast, the formation of cytoskeleton structures containing tubulin was unhindered in S100A10 depleted cells. Interestingly, the cellular distribution of annexin A2, an interaction partner of S100A10, was unaffected in S100A10 depleted cells. Cells lacking S100A10 showed an impaired migration activity and were unable to close a scratched wound. Our data provide first insights of S100A10 function as a regulator of the filamentous actin network. PMID:20100475

  20. Partial Depletion of Gamma-Actin Suppresses Microtubule Dynamics

    PubMed Central

    Po'uha, Sela T; Honore, Stephane; Braguer, Diane; Kavallaris, Maria

    2013-01-01

    Actin and microtubule interactions are important for many cellular events, however these interactions are poorly described. Alterations in γ-actin are associated with diseases such as hearing loss and cancer. Functional investigations demonstrated that partial depletion of γ-actin affects cell polarity and induces resistance to microtubule-targeted agents. To determine whether γ-actin alterations directly affect microtubule dynamics, microtubule dynamic instability was analyzed in living cells following partial siRNA depletion of γ-actin. Partial depletion of γ-actin suppresses interphase microtubule dynamics by 17.5% due to a decrease in microtubule shortening rates and an increase in microtubule attenuation. γ-Actin partial depletion also increased distance-based microtubule catastrophe and rescue frequencies. In addition, knockdown of γ-actin delayed mitotic progression, partially blocking metaphase–anaphase transition and inhibiting cell proliferation. Interestingly, in the presence of paclitaxel, interphase microtubule dynamics were further suppressed by 24.4% in the γ-actin knockdown cells, which is comparable to 28.8% suppression observed in the control siRNA treated cells. Paclitaxel blocked metaphase–anaphase transition in both the γ-actin knockdown cells and the control siRNA cells. However, the extent of mitotic arrest was much higher in the control cells (28.4%), compared to the γ-actin depleted cells (8.5%). Therefore, suppression of microtubule dynamics by partial depletion of γ-actin is associated with marked delays in metaphase-anaphase transition and not mitotic arrest. This is the first demonstration that γ-actin can modulate microtubule dynamics by reducing the microtubule shortening rate, promoting paused/attenuated microtubules, and increasing transition frequencies suggesting a mechanistic link between γ-actin and microtubules. © 2013 Wiley Periodicals, Inc PMID:23335583

  1. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011

  2. Reconstitution and Protein Composition Analysis of Endocytic Actin Patches

    PubMed Central

    Michelot, Alphée; Costanzo, Michael; Sarkeshik, Ali; Boone, Charles; Yates, John R.; Drubin, David G.

    2010-01-01

    Summary Background Clathrin-actin-mediated endocytosis in yeast involves the progressive assembly of at least 60 different proteins at cortical sites. More than half of these proteins are involved in the assembly of a branched network of actin filaments to provide the forces required for plasma membrane invagination. Results To gain insights into the regulation of endocytic actin patch dynamics, we developed an in vitro actin assembly assay using microbeads functionalized with the nucleation promoting factor (NPF) Las17 (yeast WASP). When incubated in a yeast extract, these beads assembled actin networks and a significant fraction became motile. Multi dimensional Protein Identification Technology (MudPIT) showed that the recruitment of actin binding proteins to these Las17-derived actin networks is selective. None of the proteins known to exclusively regulate the in vivo formation of actin cables or the actin contractile ring were identified. Intriguingly, our analysis also identified components of three other cortical structures, eisosomes, PIK patches and the TORC2 complex, establishing intriguing biochemical connections between four different yeast cortical complexes. Finally, we identified Aim3 as a regulator of actin dynamics at endocytic sites. Conclusions WASP is sufficient to trigger assembly of actin networks composed selectively of actin-patch proteins. These experiments establish that the protein composition of different F-actin structures is determined by the protein factor that initiates the network. The identification of binding partners revealed new biochemical connections between WASP derived networks and other cortical complexes and identified Aim3 as a novel regulator of the endocytic actin patch. PMID:21035341

  3. Caffeine relaxes smooth muscle through actin depolymerization.

    PubMed

    Tazzeo, Tracy; Bates, Genevieve; Roman, Horia Nicolae; Lauzon, Anne-Marie; Khasnis, Mukta D; Eto, Masumi; Janssen, Luke J

    2012-08-15

    Caffeine is sometimes used in cell physiological studies to release internally stored Ca(2+). We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE. Although caffeine is an agonist of bitter taste receptors, which in turn mediate bronchodilation, its relaxant effect was not mimicked by quinine. After permeabilizing the membrane using β-escin and depleting the internal Ca(2+) store using A23187, we found that 10 mM caffeine reversed tone evoked by direct application of Ca(2+), suggesting it functionally antagonizes the contractile apparatus. Using a variety of molecular techniques, we found that caffeine did not affect phosphorylation of myosin light chain (MLC) by MLC kinase, actin-filament motility catalyzed by MLC kinase, phosphorylation of CPI-17 by either protein kinase C or RhoA kinase, nor the activity of MLC-phosphatase. However, we did obtain evidence that caffeine decreased actin filament binding to phosphorylated myosin heads and increased the ratio of globular to filamentous actin in precontracted tissues. We conclude that, in addition to its other non-RyR targets, caffeine also interferes with actin function (decreased binding by myosin, possibly with depolymerization), an effect that should be borne in mind in studies using caffeine to probe excitation-contraction coupling in smooth muscle.

  4. Summertime actinic lichenoid eruption (lichen planus actinicus).

    PubMed

    Isaacson, D; Turner, M L; Elgart, M L

    1981-04-01

    A patient with an unusual lichenoid photosensitive eruption is presented. The features differentiating this entity from classic lichen planus, lupus erythematosus, and polymorphous light eruption are discussed. Phototesting confirmed the provocative influence of sunlight. We prefer the name "summertime actinic lichenoid eruption" to that of "lichen planus subtropicus" or "lichen planus actinicus," since this condition is not confined to subtropical countries, and, although light appears to initiate the eruption, its exact relationship to lichen planus is unclear.

  5. Viscoelastic properties of actin-coated membranes.

    PubMed

    Helfer, E; Harlepp, S; Bourdieu, L; Robert, J; MacKintosh, F C; Chatenay, D

    2001-02-01

    In living cells, cytoskeletal filaments interact with the plasma membrane to form structures that play a key role in cell shape and mechanical properties. To study the interaction between these basic components, we designed an in vitro self-assembled network of actin filaments attached to the outer surface of giant unilamellar vesicles. Optical tweezers and single-particle tracking experiments are used to study the rich dynamics of these actin-coated membranes (ACM). We show that microrheology studies can be carried out on such an individual microscopic object. The principle of the experiment consists in measuring the thermally excited position fluctuations of a probe bead attached biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin network modifies strongly the membrane dynamics with respect to a fluid, lipid bilayer one. It induces first a finite (omega=0) two-dimensional (2D) shear modulus G(0)(2D) approximately 0.5 to 5 microN/m in the membrane plane. Moreover, the frequency dependence at high frequency of the shear modulus [G(')(2D)(f ) approximately f(0.85+/-0.07)] and of the bending modulus (kappa(ACM)(f) approximately f(0.55+/-0.21)) demonstrate the viscoelastic behavior of the composite membrane. These results are consistent with a common exponent of 0.75 for both moduli as expected from our model and from prior measurements on actin solutions.

  6. Novel roles for actin in mitochondrial fission

    PubMed Central

    Hatch, Anna L.; Gurel, Pinar S.; Higgs, Henry N.

    2014-01-01

    ABSTRACT Mitochondrial dynamics, including fusion, fission and translocation, are crucial to cellular homeostasis, with roles in cellular polarity, stress response and apoptosis. Mitochondrial fission has received particular attention, owing to links with several neurodegenerative diseases. A central player in fission is the cytoplasmic dynamin-related GTPase Drp1, which oligomerizes at the fission site and hydrolyzes GTP to drive membrane ingression. Drp1 recruitment to the outer mitochondrial membrane (OMM) is a key regulatory event, which appears to require a pre-constriction step in which the endoplasmic reticulum (ER) and mitochondrion interact extensively, a process termed ERMD (ER-associated mitochondrial division). It is unclear how ER–mitochondrial contact generates the force required for pre-constriction or why pre-constriction leads to Drp1 recruitment. Recent results, however, show that ERMD might be an actin-based process in mammals that requires the ER-associated formin INF2 upstream of Drp1, and that myosin II and other actin-binding proteins might be involved. In this Commentary, we present a mechanistic model for mitochondrial fission in which actin and myosin contribute in two ways; firstly, by supplying the force for pre-constriction and secondly, by serving as a coincidence detector for Drp1 binding. In addition, we discuss the possibility that multiple fission mechanisms exist in mammals. PMID:25217628

  7. Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility

    PubMed Central

    Benanti, Erin L.; Nguyen, Catherine M.; Welch, Matthew D.

    2015-01-01

    Summary Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, while their close relative B. thailandensis is nonpathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection. PMID:25860613

  8. Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility.

    PubMed

    Benanti, Erin L; Nguyen, Catherine M; Welch, Matthew D

    2015-04-01

    Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, whereas their close relative B. thailandensis is non-pathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion, and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate, and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection.

  9. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 PMID:26295568

  10. High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label.

    PubMed

    Binder, Benjamin P; Cornea, Sinziana; Thompson, Andrew R; Moen, Rebecca J; Thomas, David D

    2015-06-30

    Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1° accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2° accuracy. We used this approach to examine the crucial ADP release step in myosin's catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures. PMID:26056276

  11. Nodal signaling regulates endodermal cell motility and actin dynamics via Rac1 and Prex1

    PubMed Central

    Housley, Michael P.; Weiner, Orion D.

    2012-01-01

    Embryo morphogenesis is driven by dynamic cell behaviors, including migration, that are coordinated with fate specification and differentiation, but how such coordination is achieved remains poorly understood. During zebrafish gastrulation, endodermal cells sequentially exhibit first random, nonpersistent migration followed by oriented, persistent migration and finally collective migration. Using a novel transgenic line that labels the endodermal actin cytoskeleton, we found that these stage-dependent changes in migratory behavior correlated with changes in actin dynamics. The dynamic actin and random motility exhibited during early gastrulation were dependent on both Nodal and Rac1 signaling. We further identified the Rac-specific guanine nucleotide exchange factor Prex1 as a Nodal target and showed that it mediated Nodal-dependent random motility. Reducing Rac1 activity in endodermal cells caused them to bypass the random migration phase and aberrantly contribute to mesodermal tissues. Together, our results reveal a novel role for Nodal signaling in regulating actin dynamics and migration behavior, which are crucial for endodermal morphogenesis and cell fate decisions. PMID:22945937

  12. AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption.

    PubMed

    Whiting, Jennifer L; Ogier, Leah; Forbush, Katherine A; Bucko, Paula; Gopalan, Janani; Seternes, Ole-Morten; Langeberg, Lorene K; Scott, John D

    2016-07-26

    Filtration through the kidney eliminates toxins, manages electrolyte balance, and controls water homeostasis. Reabsorption of water from the luminal fluid of the nephron occurs through aquaporin-2 (AQP2) water pores in principal cells that line the kidney-collecting duct. This vital process is impeded by formation of an "actin barrier" that obstructs the passive transit of AQP2 to the plasma membrane. Bidirectional control of AQP2 trafficking is managed by hormones and signaling enzymes. We have discovered that vasopressin-independent facets of this homeostatic mechanism are under the control of A-Kinase Anchoring Protein 220 (AKAP220; product of the Akap11 gene). CRISPR/Cas9 gene editing and imaging approaches show that loss of AKAP220 disrupts apical actin networks in organoid cultures. Similar defects are evident in tissue sections from AKAP220-KO mice. Biochemical analysis of AKAP220-null kidney extracts detected reduced levels of active RhoA GTPase, a well-known modulator of the actin cytoskeleton. Fluorescent imaging of kidney sections from these genetically modified mice revealed that RhoA and AQP2 accumulate at the apical surface of the collecting duct. Consequently, these animals are unable to appropriately dilute urine in response to overhydration. We propose that membrane-proximal signaling complexes constrained by AKAP220 impact the actin barrier dynamics and AQP2 trafficking to ensure water homeostasis. PMID:27402760

  13. Localizations of γ-Actins in Skin, Hair, Vibrissa, Arrector Pili Muscle and Other Hair Appendages of Developing Rats

    PubMed Central

    Morioka, Kiyokazu; Takano-Ohmuro, Hiromi

    2016-01-01

    Six isoforms of actins encoded by different genes have been identified in mammals including α-cardiac, α-skeletal, α-smooth muscle (α-SMA), β-cytoplasmic, γ-smooth muscle (γ-SMA), and γ-cytoplasmic actins (γ-CYA). In a previous study we showed the localization of α-SMA and other cytoskeletal proteins in the hairs and their appendages of developing rats (Morioka K., et al. (2011) Acta Histochem. Cytochem. 44, 141–153), and herein we determined the localization of γ type actins in the same tissues and organs by immunohistochemical staining. Our results indicate that the expression of γ-SMA and γ-CYA is suggested to be poor in actively proliferating tissues such as the basal layer of the epidermis and the hair matrix in the hair bulb, and as well as in highly keratinized tissues such as the hair cortex and hair cuticle. In contrast, the expression of γ-actins were high in the spinous layer, granular layer, hair shaft, and inner root sheath, during their active differentiations. In particular, the localization of γ-SMA was very similar to that of α-SMA. It was located not only in the arrector pili muscles and muscles in the dermis, but also in the dermal sheath and in a limited area of the outer root sheath in both the hair and vibrissal follicles. The γ-CYA was suggested to be co-localized with γ-SMA in the dermal sheath, outer root sheath, and arrector pili muscles. Sparsely distributed dermal cells expressed both types of γ-actin. The expression of γ-actins is suggested to undergo dynamic changes according to the proliferation and differentiation of the skin and hair-related cells. PMID:27222613

  14. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana

    PubMed Central

    Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1–actin complex, we constructed a homology model of the AtADF1–actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson–Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  15. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

    PubMed

    Du, Juan; Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  16. Actin Filaments at the Leading Edge of Cancer Cells Are Characterized by a High Mobile Fraction and Turnover Regulation by Profilin I

    PubMed Central

    Lorente, Gisela; Syriani, Emilio; Morales, Miguel

    2014-01-01

    Cellular motility is the basis for cancer cell invasion and metastasis. In the case of breast cancer, the most common type of cancer among women, metastasis represents the most devastating stage of the disease. The central role of cellular motility in cancer development emphasizes the importance of understanding the specific mechanisms involved in this process. In this context, tumor development and metastasis would be the consequence of a loss or defect of the mechanisms that control cytoskeletal remodeling. Profilin I belongs to a family of small actin binding proteins that are thought to assist in actin filament elongation at the leading edge of migrating cells. Traditionally, Profilin I has been considered to be an essential control element for actin polymerization and cell migration. Expression of Profilin I is down-regulated in breast and various other cancer cells. In MDA-MB-231 cells, a breast cancer cell line, further inhibition of Profilin I expression promotes hypermotility and metastatic spread, a finding that contrasts with the proposed role of Profilin in enhancing polymerization. In this report, we have taken advantage of the fluorescence recovery after photobleaching (FRAP) of GFP-actin to quantify and compare actin dynamics at the leading edge level in both cancer and non-cancer cell models. Our results suggest that (i) a high level of actin dynamics (i.e., a large mobile fraction of actin filaments and a fast turnover) is a common characteristic of some cancer cells; (ii) actin polymerization shows a high degree of independence from the presence of extracellular growth factors; and (iii) our results also corroborate the role of Profilin I in regulating actin polymerization, as raising the intracellular levels of Profilin I decreased the mobile fraction ratio of actin filaments and slowed their polymerization rate; furthermore, increased Profilin levels also led to reduced individual cell velocity and directionality. PMID:24465723

  17. Actin is required for IFT regulation in Chlamydomonas reinhardtii.

    PubMed

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C; Sale, Winfield S; Shoichet, Brian; Pringle, John R; Marshall, Wallace F

    2014-09-01

    Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.

  18. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

  19. The neuronal and actin commitment: Why do neurons need rings?

    PubMed

    Leite, Sérgio Carvalho; Sousa, Mónica Mendes

    2016-09-01

    The role of the actin cytoskeleton in neurons has been extensively studied in actin-enriched compartments such as the growth cone and dendritic spines. The recent discovery of actin rings in the axon shaft and in dendrites, together with the identification of axon actin trails, has advanced our understanding on actin organization and dynamics in neurons. However, specifically in the case of actin rings, the mechanisms regulating their nucleation and assembly, and the functions that they may exert in axons and dendrites remain largely unexplored. Here we discuss the possible structural, mechanistic and functional properties of the subcortical neuronal cytoskeleton putting the current knowledge in perspective with the information available on actin rings formed in other biological contexts, and with the organization of actin-spectrin lattices in other cell types. The detailed analysis of these novel neuronal actin ring structures, together with the elucidation of the function of actin-binding proteins in neuron biology, has a large potential to uncover new mechanisms of neuronal function under normal conditions that may have impact in our understanding of axon degeneration and regeneration. © 2016 Wiley Periodicals, Inc.

  20. Excitable actin dynamics in lamellipodial protrusion and retraction.

    PubMed

    Ryan, Gillian L; Petroccia, Heather M; Watanabe, Naoki; Vavylonis, Dimitrios

    2012-04-01

    Many animal cells initiate crawling by protruding lamellipodia, consisting of a dense network of actin filaments, at their leading edge. We imaged XTC cells that exhibit flat lamellipodia on poly-L-lysine-coated coverslips. Using active contours, we tracked the leading edge and measured the total amount of F-actin by summing the pixel intensities within a 5-μm band. We observed protrusion and retraction with period 130-200 s and local wavelike features. Positive (negative) velocities correlated with minimum (maximum) integrated actin concentration. Approximately constant retrograde flow indicated that protrusions and retractions were driven by fluctuations of the actin polymerization rate. We present a model of these actin dynamics as an excitable system in which a diffusive, autocatalytic activator causes actin polymerization; F-actin accumulation in turn inhibits further activator accumulation. Simulations of the model reproduced the pattern of actin polymerization seen in experiments. To explore the model's assumption of an autocatalytic activation mechanism, we imaged cells expressing markers for both F-actin and the p21 subunit of the Arp2/3 complex. We found that integrated Arp2/3-complex concentrations spike several seconds before spikes of F-actin concentration. This suggests that the Arp2/3 complex participates in an activation mechanism that includes additional diffuse components. Response of cells to stimulation by fetal calf serum could be reproduced by the model, further supporting the proposed dynamical picture.

  1. Effect of Tropomyosin on Formin-Bound Actin Filaments

    PubMed Central

    Ujfalusi, Zoltán; Vig, Andrea; Hild, Gábor; Nyitrai, Miklós

    2009-01-01

    Abstract Formins are conservative proteins with important roles in the regulation of the microfilament system in eukaryotic cells. Previous studies showed that the binding of formins to actin made the structure of actin filaments more flexible. Here, the effects of tropomyosin on formin-induced changes in actin filaments were investigated using fluorescence spectroscopic methods. The temperature dependence of the Förster-type resonance energy transfer showed that the formin-induced increase of flexibility of actin filaments was diminished by the binding of tropomyosin to actin. Fluorescence anisotropy decay measurements also revealed that the structure of flexible formin-bound actin filaments was stabilized by the binding of tropomyosin. The stabilizing effect reached its maximum when all binding sites on actin were occupied by tropomyosin. The effect of tropomyosin on actin filaments was independent of ionic strength, but became stronger as the magnesium concentration increased. Based on these observations, we propose that in cells there is a molecular mechanism in which tropomyosin binding to actin plays an important role in forming mechanically stable actin filaments, even in the case of formin-induced rapid filament assembly. PMID:18931257

  2. The neuronal and actin commitment: Why do neurons need rings?

    PubMed

    Leite, Sérgio Carvalho; Sousa, Mónica Mendes

    2016-09-01

    The role of the actin cytoskeleton in neurons has been extensively studied in actin-enriched compartments such as the growth cone and dendritic spines. The recent discovery of actin rings in the axon shaft and in dendrites, together with the identification of axon actin trails, has advanced our understanding on actin organization and dynamics in neurons. However, specifically in the case of actin rings, the mechanisms regulating their nucleation and assembly, and the functions that they may exert in axons and dendrites remain largely unexplored. Here we discuss the possible structural, mechanistic and functional properties of the subcortical neuronal cytoskeleton putting the current knowledge in perspective with the information available on actin rings formed in other biological contexts, and with the organization of actin-spectrin lattices in other cell types. The detailed analysis of these novel neuronal actin ring structures, together with the elucidation of the function of actin-binding proteins in neuron biology, has a large potential to uncover new mechanisms of neuronal function under normal conditions that may have impact in our understanding of axon degeneration and regeneration. © 2016 Wiley Periodicals, Inc. PMID:26784007

  3. The Yeast Actin Cytoskeleton: from Cellular Function to Biochemical Mechanism

    PubMed Central

    Moseley, James B.; Goode, Bruce L.

    2006-01-01

    All cells undergo rapid remodeling of their actin networks to regulate such critical processes as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. These events are driven by the coordinated activities of a set of 20 to 30 highly conserved actin-associated proteins, in addition to many cell-specific actin-associated proteins and numerous upstream signaling molecules. The combined activities of these factors control with exquisite precision the spatial and temporal assembly of actin structures and ensure dynamic turnover of actin structures such that cells can rapidly alter their cytoskeletons in response to internal and external cues. One of the most exciting principles to emerge from the last decade of research on actin is that the assembly of architecturally diverse actin structures is governed by highly conserved machinery and mechanisms. With this realization, it has become apparent that pioneering efforts in budding yeast have contributed substantially to defining the universal mechanisms regulating actin dynamics in eukaryotes. In this review, we first describe the filamentous actin structures found in Saccharomyces cerevisiae (patches, cables, and rings) and their physiological functions, and then we discuss in detail the specific roles of actin-associated proteins and their biochemical mechanisms of action. PMID:16959963

  4. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition. PMID:26240174

  5. Sequence and comparative genomic analysis of actin-related proteins.

    PubMed

    Muller, Jean; Oma, Yukako; Vallar, Laurent; Friederich, Evelyne; Poch, Olivier; Winsor, Barbara

    2005-12-01

    Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4.

  6. Dissociation of F-actin induced by hydrostatic pressure.

    PubMed

    Garcia, C R; Amaral Júnior, J A; Abrahamsohn, P; Verjovski-Almeida, S

    1992-11-01

    F-actin purified from rabbit skeletal muscle undergoes reversible dissociation when subjected to hydrostatic pressures up to 240 MPa. Dissociation and reversibility were detected by the following procedures: fluorescence spectral changes observed under pressure, when either intrinsic tryptophan or pyrenyl emission of N-(1-pyrenyl)iodoacetamide-labeled actin were monitored; electron microscopy of samples fixed under pressure; size-exclusion HPLC of pressurized actin. The effect of pressure upon F-actin that had been polymerized in the presence of either Mg2+, Ca2+ or K+ was studied. The standard volume changes for the association of actin subunits, calculated from pressure/dissociation curves were 74 +/- 14 ml/mol for Mg-F-actin, 79 +/- 12 ml/mol for Ca-F-actin and 328 +/- 63 ml/mol for K-F-actin, indicating that actin subunits are packed differently in the polymer depending on which cation is present. All pressure/dissociation data could be fitted by a model for dissociation of a dimer, which suggests that in the F-actin filament there is a predominant intersubunit interaction interface, most likely the head-to-tail intrastrand interaction between two subunits which repeats itself along the polymer. A tenfold change in total protein concentration from 20 micrograms to 200 micrograms/ml Mg-F-actin did not cause a change in the pressure required for half-maximal dissociation. This indicates a heterogeneity of free energy of association among actin monomers in the Mg-F-actin polymer, suggesting that, in addition to the predominant intersubunit interaction, the disordered interactions in the filament significantly contribute to the heterogeneity of microenvironments in the interface between the subunits. PMID:1425683

  7. Fibroblast-mediated contraction in actinically exposed and actinically protected aging skin

    SciTech Connect

    Marks, M.W.; Morykwas, M.J.; Wheatley, M.J. )

    1990-08-01

    The changes in skin morphology over time are a consequence of both chronologic aging and the accumulation of environmental exposure. Through observation, we know that actinic radiation intensifies the apparent aging of skin. We have investigated the effects of aging and actinic radiation on the ability of fibroblasts to contract collagen-fibroblast lattices. Preauricular and postauricular skin samples were obtained from eight patients aged 49 to 74 undergoing rhytidectomy. The samples were kept separate, and the fibroblasts were grown in culture. Lattices constructed with preauricular fibroblasts consistently contracted more than lattices containing postauricular fibroblasts. The difference in amount of contraction in 7 days between sites was greatest for the younger patients and decreased linearly as donor age increased (r = -0.96). This difference may be due to preauricular fibroblasts losing their ability to contract a lattice as aging skin is exposed to more actinic radiation.

  8. Photorhabdus luminescens toxins ADP-ribosylate actin and RhoA to force actin clustering.

    PubMed

    Lang, Alexander E; Schmidt, Gudula; Schlosser, Andreas; Hey, Timothy D; Larrinua, Ignacio M; Sheets, Joel J; Mannherz, Hans G; Aktories, Klaus

    2010-02-26

    The bacterium Photorhabdus luminescens is mutualistically associated with entomopathogenetic nematodes. These nematodes invade insect larvae and release the bacteria from their intestine, which kills the insects through the action of toxin complexes. We elucidated the mode of action of two of these insecticidal toxins from P. luminescens. We identified the biologically active components TccC3 and TccC5 as adenosine diphosphate (ADP)-ribosyltransferases, which modify unusual amino acids. TccC3 ADP-ribosylated threonine-148 of actin, resulting in actin polymerization. TccC5 ADP-ribosylated Rho guanosine triphosphatase proteins at glutamine-61 and glutamine-63, inducing their activation. The concerted action of both toxins inhibited phagocytosis of target insect cells and induced extensive intracellular polymerization and clustering of actin. Several human pathogenic bacteria produce related toxins. PMID:20185726

  9. Actin-Dynamics in Plant Cells: The Function of Actin Perturbing Substances Jasplakinolide, Chondramides, Phalloidin, Cytochalasins, and Latrunculins

    PubMed Central

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

    This chapter will give an overview of the most common F-actin perturbing substances, that are used to study actin dynamics in living plant cells in studies on morphogenesis, motility, organelle movement or when apoptosis has to be induced. These substances can be divided into two major subclasses – F-actin stabilizing and polymerizing substances like jasplakinolide, chondramides and F-actin severing compounds like chytochalasins and latrunculins. Jasplakinolide was originally isolated form a marine sponge, and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane permeable F-actin stabilizing and polymerizing agent, which may even have anti-cancer activities. Cytochalasins, derived from fungi show an F-actin severing function and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from red sea sponges, however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide a stable complex with actin dimers is formed resulting also in severing of F-actin. For influencing F-actin dynamics in plant cells only membrane permeable drugs are useful in a broad range. We however introduce also the phallotoxins and synthetic derivatives, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes, but has also been described in siphonal giant algae. In the present chapter the focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; however methods by fluorescence applications including phalloidin- and antibody staining as well as immunofluorescence-localization of the inhibitor drugs are given. PMID:26498789

  10. Reorganized actin filaments anchor chloroplasts along the anticlinal walls of Vallisneria epidermal cells under high-intensity blue light.

    PubMed

    Sakai, Yuuki; Takagi, Shingo

    2005-08-01

    In epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light (BL) induces the avoidance response of chloroplasts. We examined simultaneous BL-induced changes in the configuration of actin filaments in the cytoplasmic layers that face the outer periclinal wall (P side) and the anticlinal wall (A side). The results clearly showed that dynamic reorganization of the actin cytoskeleton occurs on both sides. Upon BL irradiation, thick, long bundles of actin filaments appeared, concomitant with the directed migration of chloroplasts from the P side to the A side. After 15-20 min of BL irradiation, fine actin bundles on only the A side appeared to associate with chloroplasts that had migrated from the P side. To examine the role of the fine actin bundles, we evaluated the anchorage of chloroplasts by centrifuging living cells. Upon BL irradiation, the resistance of chloroplasts on both the P and A sides to the centrifugal force decreased remarkably. After 20 min of BL irradiation, the resistance of chloroplasts on the A side increased again, but chloroplasts on the P side could still be displaced. The BL-induced recovery of resistance of chloroplasts on the A side was sensitive to photosynthesis inhibitors but insensitive to an inhibitor of flavoproteins. The photosynthesis inhibitors also prevented the fine actin bundles from appearing on the A side under BL irradiation. These results strongly suggest that the BL-induced avoidance response of chloroplasts includes photosynthesis-dependent and actin-dependent anchorage of chloroplasts on the A side of epidermal cells. PMID:15809866

  11. Spatial control of actin polymerization during neutrophil chemotaxis

    PubMed Central

    Weiner, Orion D.; Servant, Guy; Welch, Matthew D.; Mitchison, Timothy J.; Sedat, John W.; Bourne, Henry R.

    2010-01-01

    Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients. PMID:10559877

  12. Reliable amplification of actin genes facilitates deep-level phylogeny.

    PubMed

    Voigt, K; Wöstemeyer, J

    2000-09-01

    The gene for actin as a highly conserved and functionally essential genetic element is developing into a major tool for phylogenetic analysis within a broad organismic range. We therefore propose a set of universally applicable primers that allow reliable amplification of actin genes. For primer construction the amino acid sequences of 57 actin genes comprising fungi, animals, plants and protists were analysed, aligned and used for the definition of six well-conserved regions which are suitable as priming sites in PCR amplification experiments. Ten primers were designed for specific in vitro amplification of actin gene fragments from a wide range of microorganisms. The corresponding gene fragments provide a strong basis to isolate nearly complete actin genes for further molecular characterization and for establishing phylogenies based on actin gene trees.

  13. Actin Depolymerization Drives Actomyosin Ring Contraction during Budding Yeast Cytokinesis

    PubMed Central

    Pinto, Inês Mendes; Rubinstein, Boris; Kucharavy, Andrei; Unruh, Jay R.; Li, Rong

    2012-01-01

    SUMMARY Actin filaments and myosin-II are evolutionarily conserved force generating components of the contractile ring during cytokinesis. Here we show that in budding yeast actin filament depolymerization plays a major role in actomyosin ring constriction. Cofilin mutation or chemically stabilizing actin filaments attenuates actomyosin ring constriction. Deletion of myosin-II motor domain or the myosin regulatory light chain reduced the contraction rate and also the rate of actin depolymerization in the ring. We constructed a quantitative microscopic model of actomyosin ring constriction via filament sliding driven by both actin depolymerization and myosin-II motor activity. Model simulations based on experimental measurements supports the notion that actin depolymerization is the predominant mechanism for ring constriction. The model predicts invariability of total contraction time irrespective of the initial ring size as originally reported for C elegans embryonic cells. This prediction was validated in yeast cells of different sizes due to having different ploidies. PMID:22698284

  14. Hybrid time dependent/independent solution for the He I line ratio temperature and density diagnostic for a thermal helium beam with applications in the scrape-off layer-edge regions in tokamaks

    SciTech Connect

    Munoz Burgos, J. M.; Schmitz, O.; Loch, S. D.; Ballance, C. P.

    2012-01-15

    Spectroscopic studies of line emission intensities and ratios offer an attractive option in the development of non-invasive plasma diagnostics. Evaluating ratios of selected He I line emission profiles from the singlet and triplet neutral helium spin systems allows for simultaneous measurement of electron density (n{sub e}) and temperature (T{sub e}) profiles. Typically, this powerful diagnostic tool is limited by the relatively long relaxation times of the {sup 3}S metastable term of helium that populates the triplet spin system, and on which electron temperature sensitive lines are based. By developing a time dependent analytical solution, we model the time evolution of the two spin systems. We present a hybrid time dependent/independent line ratio solution that improves the range of application of this diagnostic technique in the scrape-off layer (SOL) and edge plasma regions when comparing it against the current equilibrium line ratio helium model used at TEXTOR.

  15. Fast two-stream method for computing diurnal-mean actinic flux in vertically inhomogeneous atmospheres

    NASA Technical Reports Server (NTRS)

    Filyushkin, V. V.; Madronich, S.; Brasseur, G. P.; Petropavlovskikh, I. V.

    1994-01-01

    Based on a derivation of the two-stream daytime-mean equations of radiative flux transfer, a method for computing the daytime-mean actinic fluxes in the absorbing and scattering vertically inhomogeneous atmosphere is suggested. The method applies direct daytime integration of the particular solutions of the two-stream approximations or the source functions. It is valid for any duration of period of averaging. The merit of the method is that the multiple scattering computation is carried out only once for the whole averaging period. It can be implemented with a number of widely used two-stream approximations. The method agrees with the results obtained with 200-point multiple scattering calculations. The method was also tested in runs with a 1-km cloud layer with optical depth of 10, as well as with aerosol background. Comparison of the results obtained for a cloud subdivided into 20 layers with those obtained for a one-layer cloud with the same optical parameters showed that direct integration of particular solutions possesses an 'analytical' accuracy. In the case of the source function interpolation, the actinic fluxes calculated above the one-layer and 20-layer clouds agreed within 1%-1.5%, while below the cloud they may differ up to 5% (in the worst case). The ways of enhancing the accuracy (in a 'two-stream sense') and computational efficiency of the method are discussed.

  16. Novel muscle-specific enhancer sequences upstream of the cardiac actin gene.

    PubMed Central

    Biben, C; Kirschbaum, B J; Garner, I; Buckingham, M

    1994-01-01

    A DNase I-hypersensitive site analysis of the 5'-flanking region of the mouse alpha-cardiac actin gene with muscle cell lines derived from C3H mice shows the presence of two such sites, at about -5 and -7 kb. When tested for activity in cultured cells with homologous and heterologous promoters, both sequences act as muscle-specific enhancers. Transcription from the proximal promoter of the alpha-cardiac actin gene is increased 100-fold with either enhancer. The activity of the distal enhancer in C2/7 myotubes is confined to an 800-bp fragment, which contains multiple E boxes. In transfection assays, this sequence does not give detectable transactivation by any of the myogenic factors even though one of the E boxes is functionally important. Bandshift assays showed that MyoD and myogenin can bind to this E box. However, additional sequences are also required for activity. We conclude that in the case of this muscle enhancer, myogenic factors alone are not sufficient to activate transcription either directly via an E box or indirectly through activation of genes encoding other muscle factors. In BALB/c mice, in which cardiac actin mRNA levels are 8- to 10-fold lower, the alpha-cardiac actin locus is perturbed by a 9.5-kb insertion (I. Garner, A. J. Minty, S. Alonso, P. J. Barton, and M. E. Buckingham, EMBO J. 5:2559-2567, 1986). This is located at -6.5 kb, between the two enhancers. The insertion therefore distances the distal enhancer from the promoter and from the proximal enhancer of the bona fide cardiac actin gene, probably thus perturbing transcriptional activity. Images PMID:8164695

  17. Expression of a dynamin 2 mutant associated with Charcot-Marie-Tooth disease leads to aberrant actin dynamics and lamellipodia formation.

    PubMed

    Yamada, Hiroshi; Kobayashi, Kinue; Zhang, Yubai; Takeda, Tetsuya; Takei, Kohji

    2016-08-15

    Specific mutations in dynamin 2 are linked to Charcot-Marie-Tooth disease (CMT), an inherited peripheral neuropathy. However, the effects of these mutations on dynamin function, particularly in relation to the regulation of the actin cytoskeleton remain unclear. Here, selected CMT-associated dynamin mutants were expressed to examine their role in the pathogenesis of CMT in U2OS cells. Ectopic expression of the dynamin CMT mutants 555Δ3 and K562E caused an approximately 50% decrease in serum stimulation-dependent lamellipodia formation; however, only K562E caused aberrations in the actin cytoskeleton. Immunofluorescence analysis showed that the K562E mutation resulted in the disappearance of radially aligned actin bundles and the simultaneous appearance of F-actin clusters. Live-cell imaging analyses showed F-actin polymers of decreased length assembled into immobile clusters in K562E-expressing cells. The K562E dynamin mutant colocalized with the F-actin clusters, whereas its colocalization with clathrin-coated pit marker proteins was decreased. Essentially the same results were obtained using another cell line, HeLa and NG108-15 cells. The present study is the first to show the association of dynamin CMT mutations with aberrant actin dynamics and lamellipodia, which may contribute to defective endocytosis and myelination in Schwann cells in CMT.

  18. Actin Cytoskeleton Regulation of Epithelial Mesenchymal Transition in Metastatic Cancer Cells

    PubMed Central

    Shankar, Jay; Nabi, Ivan R.

    2015-01-01

    Epithelial-mesenchymal transition (EMT) is associated with loss of the cell-cell adhesion molecule E-cadherin and disruption of cell-cell junctions as well as with acquisition of migratory properties including reorganization of the actin cytoskeleton and activation of the RhoA GTPase. Here we show that depolymerization of the actin cytoskeleton of various metastatic cancer cell lines with Cytochalasin D (Cyt D) reduces cell size and F-actin levels and induces E-cadherin expression at both the protein and mRNA level. Induction of E-cadherin was dose dependent and paralleled loss of the mesenchymal markers N-cadherin and vimentin. E-cadherin levels increased 2 hours after addition of Cyt D in cells showing an E-cadherin mRNA response but only after 10-12 hours in HT-1080 fibrosarcoma and MDA-MB-231 cells in which E-cadherin mRNA level were only minimally affected by Cyt D. Cyt D treatment induced the nuclear-cytoplasmic translocation of EMT-associated SNAI 1 and SMAD1/2/3 transcription factors. In non-metastatic MCF-7 breast cancer cells, that express E-cadherin and represent a cancer cell model for EMT, actin depolymerization with Cyt D induced elevated E-cadherin while actin stabilization with Jasplakinolide reduced E-cadherin levels. Elevated E-cadherin levels due to Cyt D were associated with reduced activation of Rho A. Expression of dominant-negative Rho A mutant increased and dominant-active Rho A mutant decreased E-cadherin levels and also prevented Cyt D induction of E-cadherin. Reduced Rho A activation downstream of actin remodelling therefore induces E-cadherin and reverses EMT in cancer cells. Cyt D treatment inhibited migration and, at higher concentrations, induced cytotoxicity of both HT-1080 fibrosarcoma cells and normal Hs27 fibroblasts, but only induced mesenchymal-epithelial transition in HT-1080 cancer cells. Our studies suggest that actin remodelling is an upstream regulator of EMT in metastatic cancer cells. PMID:25756282

  19. Correlative nanoscale imaging of actin filaments and their complexes

    PubMed Central

    Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil

    2014-01-01

    Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP–actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP–actin complexes. PMID:23727693

  20. Human cytoplasmic actin proteins are encoded by a multigene family

    SciTech Connect

    Engel, J.; Gunning, P.; Kedes, L.

    1982-06-01

    The authors characterized nine human actin genes that they isolated from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and ..cap alpha..-, ..beta..-, and ..gamma..-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria they show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken ..beta..-actin cDNA. They conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

  1. Solubilization of native actin monomers from human erythrocyte membranes.

    PubMed

    Tilley, L; Dwyer, M; Ralston, G B

    1986-01-01

    Up to 50% of the actin in erythrocyte membranes can be solubilized at low ionic strength in a form capable of inhibiting DNAse I, in the presence of 0.4 mM ATP and 0.05 mM calcium. In the absence of calcium and ATP, actin is released but is apparently rapidly denatured. Solubilization of G-actin increases with temperature up to 37 degrees C. At higher temperatures, actin is released rapidly but quickly loses its ability to inhibit DNAse I. PMID:3789986

  2. An unconventional form of actin in protozoan hemoflagellate, Leishmania.

    PubMed

    Kapoor, Prabodh; Sahasrabuddhe, Amogh A; Kumar, Ashutosh; Mitra, Kalyan; Siddiqi, Mohammad Imran; Gupta, Chhitar M

    2008-08-15

    Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs. PMID:18539603

  3. Actin in dendritic spines: connecting dynamics to function

    PubMed Central

    2010-01-01

    Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic part of most excitatory synapses and are major sites of information processing and storage in the brain. Changes in the shape and size of dendritic spines are correlated with the strength of excitatory synaptic connections and heavily depend on remodeling of its underlying actin cytoskeleton. Emerging evidence suggests that most signaling pathways linking synaptic activity to spine morphology influence local actin dynamics. Therefore, specific mechanisms of actin regulation are integral to the formation, maturation, and plasticity of dendritic spines and to learning and memory. PMID:20457765

  4. Helical buckling of actin inside filopodia generates traction

    PubMed Central

    Leijnse, Natascha; Oddershede, Lene B.; Bendix, Poul M.

    2015-01-01

    Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell–cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle. PMID:25535347

  5. Correlative nanoscale imaging of actin filaments and their complexes.

    PubMed

    Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E; Reisler, Emil; Gimzewski, James K

    2013-07-01

    Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

  6. The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells

    PubMed Central

    Yasunaga, Takayuki; Hoff, Sylvia; Schell, Christoph; Helmstädter, Martin; Kretz, Oliver; Kuechlin, Sebastian; Yakulov, Toma A.; Engel, Christina; Müller, Barbara; Bensch, Robert; Ronneberger, Olaf; Huber, Tobias B.; Lienkamp, Soeren S.

    2015-01-01

    Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton. PMID:26644512

  7. The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells.

    PubMed

    Yasunaga, Takayuki; Hoff, Sylvia; Schell, Christoph; Helmstädter, Martin; Kretz, Oliver; Kuechlin, Sebastian; Yakulov, Toma A; Engel, Christina; Müller, Barbara; Bensch, Robert; Ronneberger, Olaf; Huber, Tobias B; Lienkamp, Soeren S; Walz, Gerd

    2015-12-01

    Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.

  8. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

    PubMed

    Chen, Guokai; Hou, Zhonggang; Gulbranson, Daniel R; Thomson, James A

    2010-08-01

    Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs.

  9. Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast.

    PubMed

    Boldogh, I R; Yang, H C; Nowakowski, W D; Karmon, S L; Hays, L G; Yates, J R; Pon, L A

    2001-03-13

    The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes. Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex.

  10. Visualization of Actin Cytoskeletal Dynamics in Fixed and Live Drosophila Egg Chambers.

    PubMed

    Groen, Christopher M; Tootle, Tina L

    2015-01-01

    Visualization of actin cytoskeletal dynamics is critical for understanding the spatial and temporal regulation of actin remodeling. Drosophila oogenesis provides an excellent model system for visualizing the actin cytoskeleton. Here, we present methods for imaging the actin cytoskeleton in Drosophila egg chambers in both fixed samples by phalloidin staining and in live egg chambers using transgenic actin labeling tools.

  11. Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells

    SciTech Connect

    Johnsson, Anna-Karin; Karlsson, Roger

    2012-01-15

    Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a range of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.

  12. Ultrastructural aspects of the development of the hyaline layer and extracellular matrix lining the gastrointestinal tract in embryos and larvae of the starfish Pisaster ochraceus preserved by freeze substitution.

    PubMed

    Pang, T; Crawford, B J; Campbell, S S

    2002-02-01

    Embryos and larvae of the starfish Pisaster ochraceus are surrounded by a complex extracellular matrix (ECM) layer called the hyaline layer (HL). A similar but less well-organized ECM layer lines some regions of the larval gut. Examination of material preserved by freeze substitution shows that the HL consists of a coarse outer meshwork, a boundary layer, a supporting layer, which is divided into three sublayers, H1, H2, and H3, and an intervillus layer. The development of the HL has been studied in material preserved by freeze substitution. Development begins at fertilization when exocytosis of the cortical granules releases ECM into the perivitelline space and elevates the fertilization membrane. Shortly after, plaques of dense material with attached fibers are present on the outer surface of the egg plasmalemma. Following this, these plaques and fibers are associated with the tips of short microvilli, suggesting that they may induce microvillus formation. Next, the tips of some of the microvilli are joined by short regions of the H1 sublayer. Some of these H1 regions have short segments of boundary layer material associated with their outer surfaces while others are naked. Just prior to hatching, the H1 and boundary layers completely surround the embryo, separating the developing coarse meshwork and intervillus layers. Short segments of the H2 and H3 sublayers are also present. Posthatching, the microvilli and all HL layers increase in thickness and density, particularly the H2, boundary, and coarse outer meshwork layers. The results suggest a sequential organization of HL components from ECM that is secreted into the perivitelline space. PMID:11748701

  13. A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface*

    PubMed Central

    Wong, Wilson; Webb, Andrew I.; Olshina, Maya A.; Infusini, Giuseppe; Tan, Yan Hong; Hanssen, Eric; Catimel, Bruno; Suarez, Cristian; Condron, Melanie; Angrisano, Fiona; NebI, Thomas; Kovar, David R.; Baum, Jake

    2014-01-01

    Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing. PMID:24371134

  14. DNA Binding Properties of the Actin-Related Protein Arp8 and Its Role in DNA Repair

    PubMed Central

    Murakami, Hirokazu; Otawa, Kenji; Tachiwana, Hiroaki; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-ich; Kurumizaka, Hitoshi; Harata, Masahiko

    2014-01-01

    Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not γ-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair. PMID:25299602

  15. HAP1 helps to regulate actin-based transport of insulin-containing granules in pancreatic β cells.

    PubMed

    Wang, Zhiyong; Peng, Ting; Wu, Hongnian; He, Jun; Li, He

    2015-07-01

    Huntingtin-associated protein 1 (HAP1) is enriched in neurons and binds to polyglutamine-expanded huntingtin. It consists of two alternatively spliced isoforms, HAP1A and HAP1B, which differ only in their short C-terminal sequences. Both HAP1A and HAP1B have been also detected in pancreatic β cells, where the loss of HAP1 impairs glucose-stimulated insulin secretion. Here, we use time-lapse laser scanning confocal microscopy to provide direct evidence that HAP1A, but not HAP1B, co-localizes and co-migrates with insulin-containing vesicles and actin-based myosin Va motor protein in the INS-1 pancreatic β cell line. Knocking down HAP1 expression using small interfering RNA significantly inhibited actin-based transport of insulin vesicles following glucose stimulation. Co-immunoprecipitation experiments demonstrated interaction between HAP1A, myosin Va, and phogrin, a transmembrane protein in insulin-containing vesicles. Stimulating INS-1 cells with glucose increased the association of HAP1A with myosin Va, while silencing HAP1 expression reduced the association of myosin Va with phogrin after glucose stimulation, without affecting levels of myosin Va or actin. Our results provide real-time evidence in living cells that HAP1 may help regulate transport of insulin-containing secretory granules along cortical actin filaments. This also raises the possibility that HAP1 may play an important role in actin-based secretory vesicle trafficking in neurons. PMID:25744490

  16. Actinic EUV mask inspection beyond 0.25 NA

    SciTech Connect

    Goldberg, Kenneth A.; Mochi, Iacopo; Anderson, Erik H.; Rekawa, Seno B.; Kemp, Charles D.; Huh, S.; Han, H.-S.

    2008-08-06

    Operating at EUV wavelengths, the SEMATECH Berkeley Actinic Inspection Tool (AIT) is a zoneplate microscope that provides high quality aerial image measurements in routine operations for SEMATECH member companies. We have upgraded the optical performance of the AIT to provide multiple image magnifications, and several inspection NA values up to 0.35 NA equivalent (0.0875 mask-side). We report on the improved imaging capabilities including resolution below 100-nm on the mask side (25 nm, 4x wafer equivalent). EUV reticles are intricate optical systems made from of several materials with wavelength-specific optical properties. The combined interactions of the substrate, multilayer-stack, buffer layer and absorber layer produce a reflected EUV optical field that is challenging to model accurately, and difficult to fully assess without actinic at-wavelength inspection. Understanding the aerial image from lithographic printing alone is complicated by photoresist properties. The AIT is now used to investigate mask issues such as amplitude and phase defect printability, pattern repair techniques, contamination, inspection damage, and mask architecture. The AIT has a 6{sup o} illumination angle, and high-resolution exposure times are typically 20 seconds per image. The AIT operates semi-automatically capturing through-focus imaging series with step sizes as small as 0.1 {micro}m (0.5-0.8 {micro}m are typical), and a step resolution of 0.05 {micro}m. We believe it is the most advanced EUV mask inspection tool in operation today. In the AIT, an EUV image of the mask is projected by a zoneplate lens with high magnification (680-910x) onto a CCD camera. The CCD over-samples the image, providing equivalent pixel sizes down to 15 nm in mask coordinates-several image pixels per resolution element. The original AIT zoneplate specifications were designed to emulate the resolution of a 0.25-NA 4x stepper, and thorough benchmarking analysis of the aberrations, flare, contrast

  17. A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

    PubMed Central

    Bonello, Teresa T.; Janco, Miro; Hook, Jeff; Byun, Alex; Appaduray, Mark; Dedova, Irina; Hitchcock-DeGregori, Sarah; Hardeman, Edna C.; Stehn, Justine R.; Böcking, Till; Gunning, Peter W.

    2016-01-01

    The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development. PMID:26804624

  18. CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor.

    PubMed

    Zhao, Shuangshuang; Jiang, Yuxiang; Zhao, Yang; Huang, Shanjin; Yuan, Ming; Zhao, Yanxiu; Guo, Yan

    2016-06-01

    The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF. PMID:27268429

  19. The effect of actin disrupting agents on contact guidance of human embryonic stem cells

    PubMed Central

    Gerecht, Sharon; Bettinger, Christopher J.; Zhang, Zhitong; Borenstein, Jeffrey; Vunjak-Novakovic, Gordana; Langer, Robert

    2007-01-01

    Mammalian cells respond to their substrates by complex changes in gene expression profiles, morphology, proliferation and migration. We report that substrate nanotopography alters morpohology and proliferation of human embryonic stem cells (hESCs). Fibronectin-coated poly(di-methyl siloxane) substrates with line-grating (600 nm ridges with 600 nm spacing and 600 +/− 150 nm feature height) induced hESC alignment and elongation, mediated the organization of cytoskeletal components including actin, vimentin, and α-tubulin, and reduced proliferation. Spatial polarization of gamma tubulin complexes was also observed in response to nanotopography. Furthermore, the addition of actin disrupting agents attenuated the alignment and proliferative effects of nanotopography. These findings further demonstrate the importance of interplay between cytoskeleton and substrate interactions as a key modulator of morphological and proliferative cellular response in hESCs on nanotopography. PMID:17576011

  20. Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity in Chlamydomonas.

    PubMed

    Onishi, Masayuki; Pringle, John R; Cross, Frederick R

    2016-03-01

    Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments ("F-actin"). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1-LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1-LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1-lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses. PMID:26715672

  1. Actin and Endocytosis in Budding Yeast

    PubMed Central

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349

  2. Encoding Mechano-Memories in Actin Networks

    NASA Astrophysics Data System (ADS)

    Foucard, Louis; Majumdar, Sayantan; Levine, Alex; Gardel, Margaret

    The ability of cells to sense and adapt to external mechanical stimuli is vital to many of its biological functions. A critical question is therefore to understand how mechanosensory mechanisms arise in living matter, with implications in both cell biology and smart materials design. Experimental work has demonstrated that the mechanical properties of semiflexible actin networks in Eukaryotic cells can be modulated (either transiently or irreversibly) via the application of external forces. Previous work has also shown with a combination of numerical simulations and analytic calculations shows that the broken rotational symmetry of the filament orientational distribution in semiflexible networks leads to dramatic changes in the mechanical response. Here we demonstrate with a combination of numerical and analytic calculations that the observed long-lived mechano-memory in the actin networks arise from changes in the nematic order of the constituent filaments. These stress-induced changes in network topology relax slowly under zero stress and can be observed through changes in the nonlinear mechanics. Our results provide a strategy for designing a novel class of materials and demonstrate a new putative mechanism of mechanical sensing in eukaryotic cells.

  3. Actin Foci Adhesion of D. discoideum

    NASA Astrophysics Data System (ADS)

    Flanders, Bret; Paneru, Govind

    2014-03-01

    Amoeboid migration is a fast (10 μm min-1) integrin-independent mode of migration that is important with D. discoideum, leukocytes, and breast cancer cells. It is poorly understood, but depends on the establishment of adhesive contacts to the substrate where the cell transmits traction forces. In pre-aggregative D. discoideum, a model system for learning about amoeboid migration, these adhesive contacts are discrete complexes that are known as actin-foci. They have an area of ~ 0.5 μm2 and a lifetime of ~ 20 s. This talk will present measurements of the adhesive character of actin foci that have been obtained using a submicron force transducer that was designed for this purpose. Results on the rupture stresses and lifetimes of individual acting foci under nano-newton level forces will be described in the context of a general theory for cellular adhesion. This theory depends on, essentially, three cellular properties: the membrane-medium surface tension, the number density of adhesion receptors in the membrane, and the receptor-substrate potential energy surface. Therefore, the use of the transducer to determine the surface tension will be presented, as well.

  4. Actin binding proteins, spermatid transport and spermiation.

    PubMed

    Qian, Xiaojing; Mruk, Dolores D; Cheng, Yan-Ho; Tang, Elizabeth I; Han, Daishu; Lee, Will M; Wong, Elissa W P; Cheng, C Yan

    2014-06-01

    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 arriving the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium beyond stage VIII of the epithelial cycle 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.

  5. Force Transmission in the Actin Cytoskeleton

    NASA Astrophysics Data System (ADS)

    Gardel, Margaret

    2012-02-01

    The ability of cells to sense and generate mechanical forces is essential to numerous aspects of their physiology, including adhesion, migration, division and differentiation. To a large degree, cellular tension is regulated by the transmission of myosin II-generated forces through the filamentous actin (F-actin) cytoskeleton. While transmission of myosin-generated stresses from the molecular to cellular length scale is well understood in the context of highly organized sarcomeres found in striated muscle, non-muscle and smooth muscle cells contain a wide variety of bundles and networks lacking sarcomeric organization. I will describe the in vitro and in vivo approaches we use to study force transmission in such disordered actomyosin assemblies. Our in vivo results are showing that highly organized stress fibers contribute surprisingly little to the overall extent of cellular tension as compared to disordered actomyosin meshworks. Our in vitro results are demonstrating the mechanisms of symmetry breaking in disordered actomyosin bundles that facilitate the formation of contractile bundles with well-defined ``contractile elements.'' These results provide insight into the self-organization of actomyosin cytoskeleton in non-muscle cells that regulate and maintain cellular tension.

  6. Mechanics of composite actin networks: in vitro and cellular perspectives

    NASA Astrophysics Data System (ADS)

    Upadhyaya, Arpita

    2014-03-01

    Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

  7. Drebrin Inhibits Cofilin-Induced Severing of F-Actin

    PubMed Central

    Grintsevich, Elena E.; Reisler, Emil

    2015-01-01

    Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer’s disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-induced severing of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor’s binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments. PMID:25047716

  8. Computational Analysis of Viscoelastic Properties of Crosslinked Actin Networks

    PubMed Central

    Kim, Taeyoon; Hwang, Wonmuk; Lee, Hyungsuk; Kamm, Roger D.

    2009-01-01

    Mechanical force plays an important role in the physiology of eukaryotic cells whose dominant structural constituent is the actin cytoskeleton composed mainly of actin and actin crosslinking proteins (ACPs). Thus, knowledge of rheological properties of actin networks is crucial for understanding the mechanics and processes of cells. We used Brownian dynamics simulations to study the viscoelasticity of crosslinked actin networks. Two methods were employed, bulk rheology and segment-tracking rheology, where the former measures the stress in response to an applied shear strain, and the latter analyzes thermal fluctuations of individual actin segments of the network. It was demonstrated that the storage shear modulus (G′) increases more by the addition of ACPs that form orthogonal crosslinks than by those that form parallel bundles. In networks with orthogonal crosslinks, as crosslink density increases, the power law exponent of G′ as a function of the oscillation frequency decreases from 0.75, which reflects the transverse thermal motion of actin filaments, to near zero at low frequency. Under increasing prestrain, the network becomes more elastic, and three regimes of behavior are observed, each dominated by different mechanisms: bending of actin filaments, bending of ACPs, and at the highest prestrain tested (55%), stretching of actin filaments and ACPs. In the last case, only a small portion of actin filaments connected via highly stressed ACPs support the strain. We thus introduce the concept of a ‘supportive framework,’ as a subset of the full network, which is responsible for high elasticity. Notably, entropic effects due to thermal fluctuations appear to be important only at relatively low prestrains and when the average crosslinking distance is comparable to or greater than the persistence length of the filament. Taken together, our results suggest that viscoelasticity of the actin network is attributable to different mechanisms depending on the amount

  9. Computational analysis of viscoelastic properties of crosslinked actin networks.

    PubMed

    Kim, Taeyoon; Hwang, Wonmuk; Lee, Hyungsuk; Kamm, Roger D

    2009-07-01

    Mechanical force plays an important role in the physiology of eukaryotic cells whose dominant structural constituent is the actin cytoskeleton composed mainly of actin and actin crosslinking proteins (ACPs). Thus, knowledge of rheological properties of actin networks is crucial for understanding the mechanics and processes of cells. We used Brownian dynamics simulations to study the viscoelasticity of crosslinked actin networks. Two methods were employed, bulk rheology and segment-tracking rheology, where the former measures the stress in response to an applied shear strain, and the latter analyzes thermal fluctuations of individual actin segments of the network. It was demonstrated that the storage shear modulus (G') increases more by the addition of ACPs that form orthogonal crosslinks than by those that form parallel bundles. In networks with orthogonal crosslinks, as crosslink density increases, the power law exponent of G' as a function of the oscillation frequency decreases from 0.75, which reflects the transverse thermal motion of actin filaments, to near zero at low frequency. Under increasing prestrain, the network becomes more elastic, and three regimes of behavior are observed, each dominated by different mechanisms: bending of actin filaments, bending of ACPs, and at the highest prestrain tested (55%), stretching of actin filaments and ACPs. In the last case, only a small portion of actin filaments connected via highly stressed ACPs support the strain. We thus introduce the concept of a 'supportive framework,' as a subset of the full network, which is responsible for high elasticity. Notably, entropic effects due to thermal fluctuations appear to be important only at relatively low prestrains and when the average crosslinking distance is comparable to or greater than the persistence length of the filament. Taken together, our results suggest that viscoelasticity of the actin network is attributable to different mechanisms depending on the amount of

  10. TSC1 controls distribution of actin fibers through its effect on function of Rho family of small GTPases and regulates cell migration and polarity.

    PubMed

    Ohsawa, Maki; Kobayashi, Toshiyuki; Okura, Hidehiro; Igarashi, Takashi; Mizuguchi, Masashi; Hino, Okio

    2013-01-01

    The tumor-suppressor genes TSC1 and TSC2 are mutated in tuberous sclerosis, an autosomal dominant multisystem disorder. The gene products of TSC1 and TSC2 form a protein complex that inhibits the signaling of the mammalian target of rapamycin complex1 (mTORC1) pathway. mTORC1 is a crucial molecule in the regulation of cell growth, proliferation and survival. When the TSC1/TSC2 complex is not functional, uncontrolled mTORC1 activity accelerates the cell cycle and triggers tumorigenesis. Recent studies have suggested that TSC1 and TSC2 also regulate the activities of Rac1 and Rho, members of the Rho family of small GTPases, and thereby influence the ensuing actin cytoskeletal organization at focal adhesions. However, how TSC1 contributes to the establishment of cell polarity is not well understood. Here, the relationship between TSC1 and the formation of the actin cytoskeleton was analyzed in stable TSC1-expressing cell lines originally established from a Tsc1-deficient mouse renal tumor cell line. Our analyses showed that cell proliferation and migration were suppressed when TSC1 was expressed. Rac1 activity in these cells was also decreased as was formation of lamellipodia and filopodia. Furthermore, the number of basal actin stress fibers was reduced; by contrast, apical actin fibers, originating at the level of the tight junction formed a network in TSC1-expressing cells. Treatment with Rho-kinase (ROCK) inhibitor diminished the number of apical actin fibers, but rapamycin had no effect. Thus, the actin fibers were regulated by the Rho-ROCK pathway independently of mTOR. In addition, apical actin fibers appeared in TSC1-deficient cells after inhibition of Rac1 activity. These results suggest that TSC1 regulates cell polarity-associated formation of actin fibers through the spatial regulation of Rho family of small GTPases.

  11. A Facile Method to Prepare Double-Layer Isoporous Hollow Fiber Membrane by In Situ Hydrogen Bond Formation in the Spinning Line.

    PubMed

    Noor, Nazia; Koll, Joachim; Radjabian, Maryam; Abetz, Clarissa; Abetz, Volker

    2016-03-01

    A double-layer hollow fiber is fabricated where an isoporous surface of polystyrene-block-poly(4-vinylpyridine) is fixed on a support layer by co-extrusion. Due to the sulfonation of the support layer material, delamination of the two layers is suppressed without increasing the number of subsequent processing steps for isoporous composite membrane formation. Electron microscope-energy-dispersive X-ray spectroscopy images unveil the existence of a high sulfur concentration in the interfacial region by which in-process H-bond formation between the layers is evidenced. For the very first time, our study reports a facile method to fabricate a sturdy isoporous double-layer hollow fiber.

  12. Bending Flexibility of Actin Filaments during Motor-Induced Sliding

    PubMed Central

    Vikhorev, Petr G.; Vikhoreva, Natalia N.; Månsson, Alf

    2008-01-01

    Muscle contraction and other forms of cell motility occur as a result of cyclic interactions between myosin molecules and actin filaments. Force generation is generally attributed to ATP-driven structural changes in myosin, whereas a passive role is ascribed to actin. However, some results challenge this view, predicting structural changes in actin during motor activity, e.g., when the actin filaments slide on a myosin-coated surface in vitro. Here, we analyzed statistical properties of the sliding filament paths, allowing us to detect changes of this type. It is interesting to note that evidence for substantial structural changes that led to increased bending flexibility of the filaments was found in phalloidin-stabilized, but not in phalloidin-free, actin filaments. The results are in accordance with the idea that a high-flexibility structural state of actin is a prerequisite for force production, but not the idea that a low-to-high flexibility transition of the actin filament should be an important component of the force-generating step per se. Finally, our data challenge the general view that phalloidin-stabilized filaments behave as native actin filaments in their interaction with myosin. This has important implications, since phalloidin stabilization is a routine procedure in most studies of actomyosin function. PMID:18835897

  13. Lateral Membrane Diffusion Modulated by a Minimal Actin Cortex

    PubMed Central

    Heinemann, Fabian; Vogel, Sven K.; Schwille, Petra

    2013-01-01

    Diffusion of lipids and proteins within the cell membrane is essential for numerous membrane-dependent processes including signaling and molecular interactions. It is assumed that the membrane-associated cytoskeleton modulates lateral diffusion. Here, we use a minimal actin cortex to directly study proposed effects of an actin meshwork on the diffusion in a well-defined system. The lateral diffusion of a lipid and a protein probe at varying densities of membrane-bound actin was characterized by fluorescence correlation spectroscopy (FCS). A clear correlation of actin density and reduction in mobility was observed for both the lipid and the protein probe. At high actin densities, the effect on the protein probe was ∼3.5-fold stronger compared to the lipid. Moreover, addition of myosin filaments, which contract the actin mesh, allowed switching between fast and slow diffusion in the minimal system. Spot variation FCS was in accordance with a model of fast microscopic diffusion and slower macroscopic diffusion. Complementing Monte Carlo simulations support the analysis of the experimental FCS data. Our results suggest a stronger interaction of the actin mesh with the larger protein probe compared to the lipid. This might point toward a mechanism where cortical actin controls membrane diffusion in a strong size-dependent manner. PMID:23561523

  14. Filament Assembly by Spire: Key Residues and Concerted Actin Binding

    PubMed Central

    Rasson, Amy S.; Bois, Justin S.; Pham, Duy Stephen L.; Yoo, Haneul; Quinlan, Margot E.

    2014-01-01

    The most recently identified class of actin nucleators, WASp Homology domain 2 (WH2) – nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or Sc), plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of Sc in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within Sc that are critical for its activity. Using this information we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that Sc binds actin filaments, in addition to monomers. PMID:25234086

  15. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins.

    PubMed

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  16. Accelerators, Brakes, and Gears of Actin Dynamics in Dendritic Spines

    PubMed Central

    Pontrello, Crystal G.; Ethell, Iryna M.

    2010-01-01

    Dendritic spines are actin-rich structures that accommodate the postsynaptic sites of most excitatory synapses in the brain. Although dendritic spines form and mature as synaptic connections develop, they remain plastic even in the adult brain, where they can rapidly grow, change, or collapse in response to normal physiological changes in synaptic activity that underlie learning and memory. Pathological stimuli can adversely affect dendritic spine shape and number, and this is seen in neurodegenerative disorders and some forms of mental retardation and autism as well. Many of the molecular signals that control these changes in dendritic spines act through the regulation of filamentous actin (F-actin), some through direct interaction with actin, and others via downstream effectors. For example, cortactin, cofilin, and gelsolin are actin-binding proteins that directly regulate actin dynamics in dendritic spines. Activities of these proteins are precisely regulated by intracellular signaling events that control their phosphorylation state and localization. In this review, we discuss how actin-regulating proteins maintain the balance between F-actin assembly and disassembly that is needed to stabilize mature dendritic spines, and how changes in their activities may lead to rapid remodeling of dendritic spines. PMID:20463852

  17. Actin puts the squeeze on Drosophila glue secretion.

    PubMed

    Merrifield, Christien J

    2016-02-01

    An actin filament coat promotes cargo expulsion from large exocytosing vesicles, but the mechanisms of coat formation and force generation have been poorly characterized. Elegant imaging studies of the Drosophila melanogaster salivary gland now reveal how actin and myosin are recruited, and show that myosin II forms a contractile 'cage' that facilitates exocytosis.

  18. Filament assembly by Spire: key residues and concerted actin binding.

    PubMed

    Rasson, Amy S; Bois, Justin S; Pham, Duy Stephen L; Yoo, Haneul; Quinlan, Margot E

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers.

  19. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins

    PubMed Central

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R.

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  20. Actin filament nucleation and elongation factors--structure-function relationships.

    PubMed

    Dominguez, Roberto

    2009-01-01

    The spontaneous and unregulated polymerization of actin filaments is inhibited in cells by actin monomer-binding proteins such as profilin and Tbeta4. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of nucleation promoting factors (NPFs), formins, Spire, Cobl, VopL/VopF, TARP and Lmod. These molecules control the time and location for polymerization, and additionally influence the structures of the actin networks that they generate. Filament nucleators are generally unrelated, but with the exception of formins they all use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs-Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. Formins are unique in that they use the formin-homology 2 (FH2) domain for interaction with actin and promote not only nucleation, but also processive barbed end elongation. In contrast, the elongation function among W-based nucleators has been "outsourced" to a dedicated family of proteins, Eva/VASP, which are related to WASP-family NPFs.

  1. Deafness and espin-actin self-organization in stereocilia

    NASA Astrophysics Data System (ADS)

    Wong, Gerard C. L.

    2009-03-01

    Espins are F-actin-bundling proteins associated with large parallel actin bundles found in hair cell stereocilia in the ear, as well as brush border microvilli and Sertoli cell junctions. We examine actin bundle structures formed by different wild-type espin isoforms, fragments, and naturally-occurring human espin mutants linked to deafness and/or vestibular dysfunction. The espin-actin bundle structure consisted of a hexagonal arrangement of parallel actin filaments in a non-native twist state. We delineate the structural consequences caused by mutations in espin's actin-bundling module. For espin mutation with a severely damaged actin-bundling module, which are implicated in deafness in mice and humans, oriented nematic-like actin filament structures, which strongly impinges on bundle mechanical stiffness. Finally, we examine what makes espin different, via a comparative study of bundles formed by espin and those formed by fascin, a prototypical bundling protein found in functionally different regions of the cell, such as filopodia.

  2. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53.

    PubMed

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1-393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using "hot-spot" p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  3. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53

    PubMed Central

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1–393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using “hot-spot” p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  4. Actin-Based Feedback Circuits in Cell Migration and Endocytosis

    NASA Astrophysics Data System (ADS)

    Wang, Xinxin

    In this thesis, we study the switch and pulse functions of actin during two important cellular processes, cell migration and endocytosis. Actin is an abundant protein that can polymerize to form a dendritic network. The actin network can exert force to push or bend the cell membrane. During cell migration, the actin network behaves like a switch, assembling mostly at one end or at the other end. The end with the majority of the actin network is the leading edge, following which the cell can persistently move in the same direction. The other end, with the minority of the actin network, is the trailing edge, which is dragged by the cell as it moves forward. When subjected to large fluctuations or external stimuli, the leading edge and the trailing edge can interchange and change the direction of motion, like a motion switch. Our model of the actin network in a cell reveals that mechanical force is crucial for forming the motion switch. We find a transition from single state symmetric behavior to switch behavior, when tuning parameters such as the force. The model is studied by both stochastic simulations, and a set of rate equations that are consistent with the simulations. Endocytosis is a process by which cells engulf extracellular substances and recycle the cell membrane. In yeast cells, the actin network is transiently needed to overcome the pressure difference across the cell membrane caused by turgor pressure. The actin network behaves like a pulse, which assembles and then disassembles within about 30 seconds. Using a stochastic model, we reproduce the pulse behaviors of the actin network and one of its regulatory proteins, Las17. The model matches green fluorescence protein (GFP) experiments for wild-type cells. The model also predicts some phenotypes that modify or diminish the pulse behavior. The phenotypes are verified with both experiments performed at Washington University and with other groups' experiments. We find that several feedback mechanisms are

  5. Interior decoration: tropomyosin in actin dynamics and cell migration.

    PubMed

    Lees, Justin G; Bach, Cuc T T; O'Neill, Geraldine M

    2011-01-01

    Cell migration and invasion requires the precise temporal and spatial orchestration of a variety of biological processes. Filaments of polymerized actin are critical players in these diverse processes, including the regulation of cell anchorage points (both cell-cell and cell-extracellular matrix), the uptake and delivery of molecules via endocytic pathways and the generation of force for both membrane protrusion and retraction. How the actin filaments are specialized for each of these discrete functions is yet to be comprehensively elucidated. The cytoskeletal tropomyosins are a family of actin associating proteins that form head-to-tail polymers which lay in the major groove of polymerized actin filaments. In the present review we summarize the emerging isoform-specific functions of tropomyosins in cell migration and invasion and discuss their potential roles in the specialization of actin filaments for the diverse cellular processes that together regulate cell migration and invasion.

  6. Actin-associated Proteins in the Pathogenesis of Podocyte Injury

    PubMed Central

    He, Fang-Fang; Chen, Shan; Su, Hua; Meng, Xian-Fang; Zhang, Chun

    2013-01-01

    Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies. PMID:24396279

  7. Side-binding proteins modulate actin filament dynamics.

    PubMed

    Crevenna, Alvaro H; Arciniega, Marcelino; Dupont, Aurélie; Mizuno, Naoko; Kowalska, Kaja; Lange, Oliver F; Wedlich-Söldner, Roland; Lamb, Don C

    2015-01-01

    Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics. PMID:25706231

  8. Role of Actin Polymerization in Cell Locomotion: Molecules and Models

    PubMed Central

    Bearer, E. L.

    2015-01-01

    Actin filaments forming at the anterior margin of a migrating cell are essential for the formation of filopodia, lamellipodia, and pseudopodia, the “feet” that the cell extends before it. These structures in turn are required for cell locomotion. Yet the molecular nature of the “nucleator” that seeds the polymerization of actin at the leading edge is unknown. Recent advances, including video microscopy of actin dynamics, discovery of proteins unique to the leading edge such as ponticulin, the Mab 2E4 antigen, and ABP 120, and novel experimental models of actin polymerization such as the actin-based movements of intracellular parasites, promise to shed light on this problem in the near future. PMID:8323743

  9. Structural Modeling and Molecular Dynamics Simulation of the Actin Filament

    SciTech Connect

    Splettstoesser, Thomas; Holmes, Kenneth; Noe, Frank; Smith, Jeremy C

    2011-01-01

    Actin is a major structural protein of the eukaryotic cytoskeleton and enables cell motility. Here, we present a model of the actin filament (F-actin) that not only incorporates the global structure of the recently published model by Oda et al. but also conserves internal stereochemistry. A comparison is made using molecular dynamics simulation of the model with other recent F-actin models. A number of structural determents such as the protomer propeller angle, the number of hydrogen bonds, and the structural variation among the protomers are analyzed. The MD comparison is found to reflect the evolution in quality of actin models over the last 6 years. In addition, simulations of the model are carried out in states with both ADP or ATP bound and local hydrogen-bonding differences characterized.

  10. Actinic prurigo of the lip: Two case reports

    PubMed Central

    Miranda, Ana MO; Ferrari, Thiago M; Werneck, Juliana T; Junior, Arley Silva; Cunha, Karin S; Dias, Eliane P

    2014-01-01

    Actinic prurigo is a photodermatosis that can affect the skin, conjunctiva and lips. It is caused by an abnormal reaction to sunlight and is more common in high-altitude living people, mainly in indigenous descendants. The diagnosis of actinic prurigo can be challenging, mainly when lip lesions are the only manifestation, which is not a common clinical presentation. The aim of this article is to report two cases of actinic prurigo showing only lip lesions. The patients were Afro-American and were unaware of possible Indian ancestry. Clinical exam, photographs, videoroscopy examination and biopsy were performed, and the diagnosis of actinic prurigo was established. Topical corticosteroid and lip balm with ultraviolet protection were prescribed with excellent results. The relevance of this report is to show that although some patients may not demonstrate the classical clinical presentation of actinic prurigo, the associated clinical and histological exams are determinants for the correct diagnosis and successful treatment of this disease. PMID:25133153

  11. How actin initiates the motor activity of Myosin.

    PubMed

    Llinas, Paola; Isabet, Tatiana; Song, Lin; Ropars, Virginie; Zong, Bin; Benisty, Hannah; Sirigu, Serena; Morris, Carl; Kikuti, Carlos; Safer, Dan; Sweeney, H Lee; Houdusse, Anne

    2015-05-26

    Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential for using the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement and may represent a strategy common to many molecular machines.

  12. Force Generation, Polymerization Dynamics and Nucleation of Actin Filaments

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

    We study force generation and actin filament dynamics using stochastic and deterministic methods. First, we treat force generation of bundled actin filaments by polymerization via molecular-level stochastic simulations. In the widely-used Brownian Ratchet model, actin filaments grow freely whenever the tip-obstacle gap created by thermal fluctuation exceeds the monomer size. We name this model the Perfect Brownian Ratchet (PBR) model. In the PBR model, actin monomer diffusion is treated implicitly. We perform a series of simulations based on the PBR, in which obstacle motion is treated explicitly; in most previous studies, obstacle motion has been treated implicitly. We find that the cooperativity of filaments is generally weak in the PBR model, meaning that more filaments would grow more slowly given the same force per filament. Closed-form formulas are also developed, which match the simulation results. These portable and accurate formulas provide guidance for experiments and upper and lower bounds for theoretical analyses. We also studied a variation of the PBR, called the Diffusing Brownian Ratchet (DBR) model, in which both actin monomer and obstacle diffusion are treated explicitly. We find that the growth rate of multiple filaments is even lower, compared with that in PBR. This finding challenges the widely-accepted PBR assumption and suggests that pushing the study of actin dynamics down to the sub-nanometer level yields new insights. We subsequently used a rate equation approach to model the effect of local depletion of actin monomers on the nucleation of actin filaments on biomimetic beads, and how the effect is regulated by capping protein (CP). We find that near the bead surface, a higher CP concentration increases local actin concentration, which leads to an enhanced activities of actin filaments' nucleation. Our model analysis matches the experimental results and lends support to an important but undervalued hypothesis proposed by Carlier and

  13. Host-cell-dependent role of actin cytoskeleton during the replication of a human strain of influenza A virus.

    PubMed

    Arcangeletti, M C; De Conto, F; Ferraglia, F; Pinardi, F; Gatti, R; Orlandini, G; Covan, S; Motta, F; Rodighiero, I; Dettori, G; Chezzi, C

    2008-01-01

    This study was aimed at investigating the possible involvement of the actin cytoskeleton in the modulation of host permissiveness to A/NWS/33 human influenza virus infection in two mammalian (MDCK and LLC-MK2) cell lines in vitro. During the early stages of infection, no appreciable association between incoming NWS/33 virions and cortical actin was detectable in the permissive MDCK model by confocal microscopy, while extensive colocalization and a slower infection progression were observed in LLC-MK2 cells. In the latter model, we also demonstrated the inability of the virus to carry out multiple replication cycles, irrespective of the presence of cleaved HA subunits in the released virions. Treatment with the actin-depolymerizing agent cytochalasin D significantly increased the infection efficiency in LLC-MK2 cells, while a detrimental effect was observed in the MDCK cell line. Our data suggest a selective role of the actin network in inducing a restriction to influenza virus replication, mostly depending on its molecular organization, the host cell type and virus replication phase. PMID:18488136

  14. A dynamic formin-dependent deep F-actin network in axons

    PubMed Central

    Ganguly, Archan; Tang, Yong; Wang, Lina; Ladt, Kelsey; Loi, Jonathan; Dargent, Bénédicte; Leterrier, Christophe

    2015-01-01

    Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles. PMID:26216902

  15. SelR/MsrB Reverses Mical-mediated Oxidation of Actin to Regulate F-actin Dynamics

    PubMed Central

    Hung, Ruei-Jiun; Spaeth, Christopher S.; Yesilyurt, Hunkar Gizem; Terman, Jonathan R.

    2014-01-01

    Actin's polymerization properties are dramatically altered by oxidation of its conserved methionine (Met)-44 residue. Mediating this effect is a specific oxidation-reduction (Redox) enzyme, Mical, that works with Semaphorin repulsive guidance cues and selectively oxidizes Met-44. We now find that this actin regulatory process is reversible. Employing a genetic approach, we identified a specific methionine sulfoxide reductase enzyme SelR that opposes Mical Redox activity and Semaphorin/Plexin repulsion to direct multiple actin-dependent cellular behaviors in vivo. SelR specifically catalyzes the reduction of the R-isomer of methionine sulfoxide (methionine-R-sulfoxide) to methionine, and we found that SelR directly reduced Mical-oxidized actin, restoring its normal polymerization properties. These results indicate that Mical oxidizes actin stereo-specifically to generate actin Met-44-R-sulfoxide (actinMet(R)O-44) – and they also implicate the interconversion of specific Met/Met(R)O residues as a precise means to modulate protein function. Our results therefore uncover a specific reversible Redox actin regulatory system that controls cell and developmental biology. PMID:24212093

  16. STED Nanoscopy of Actin Dynamics in Synapses Deep Inside Living Brain Slices

    PubMed Central

    Urban, Nicolai T.; Willig, Katrin I.; Hell, Stefan W.; Nägerl, U. Valentin

    2011-01-01

    It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. Using aberration-reducing optics, we demonstrate that it is possible to achieve stimulated emission depletion superresolution imaging deep inside scattering biological tissue. To illustrate the power of this novel (to our knowledge) approach, we resolved distinct distributions of actin inside dendrites and spines with a resolution of 60–80 nm in living organotypic brain slices at depths up to 120 μm. In addition, time-lapse stimulated emission depletion imaging revealed changes in actin-based structures inside spines and spine necks, and showed that these dynamics can be modulated by neuronal activity. Our approach greatly facilitates investigations of actin dynamics at the nanoscale within functionally intact brain tissue. PMID:21889466

  17. Human MCF10A Mammary Epithelial Cells Undergo Apoptosis following Actin Depolymerization That Is Independent of Attachment and Rescued by Bcl-2

    PubMed Central

    Martin, Stuart S.; Leder, Philip

    2001-01-01

    Many tumor cells are impaired in adhesion-regulated apoptosis, which contributes to their metastatic potential. However, suppression of this apoptotic pathway in untransformed cells is not mediated only by adhesion to the extracellular matrix but also through the resulting ability to spread and adopt a distinct morphology. Since cell spreading is dependent on the integrity of the actin microfilament cytoskeleton, we sought to determine if actin depolymerization was sufficient to induce apoptosis, even in the presence of continuous attachment. For this study, we used a human mammary epithelial cell line (MCF10A), which is immortalized but remains adhesion dependent for survival. Treatment of MCF10A cells with latrunculin-A (LA), an inhibitor of actin polymerization, rapidly led to disruption of the actin cytoskeleton and caused cell rounding but preserved attachment. Initiation of apoptosis in LA-treated MCF10A cells was detected by mitochondrial localization of the Bax apoptotic protein, which was prevented by overexpression of Bcl-2. DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage in LA-treated MCF10A cells indicated progression to the execution phase of apoptosis. The MDA-MB-453 cell line, which was derived from a metastatic human mammary tumor, was resistant to PARP cleavage and loss of viability in response to actin depolymerization. Stable overexpression of Bcl-2 in the untransformed MCF10A cells was able to recapitulate the resistance to apoptosis found in the tumor cell line. We demonstrate that inhibition of actin polymerization is sufficient to stimulate apoptosis in attached MCF10A cells, and we present a novel role for Bcl-2 in cell death induced by direct disruption of the actin cytoskeleton. PMID:11533241

  18. Human MCF10A mammary epithelial cells undergo apoptosis following actin depolymerization that is independent of attachment and rescued by Bcl-2.

    PubMed

    Martin, S S; Leder, P

    2001-10-01

    Many tumor cells are impaired in adhesion-regulated apoptosis, which contributes to their metastatic potential. However, suppression of this apoptotic pathway in untransformed cells is not mediated only by adhesion to the extracellular matrix but also through the resulting ability to spread and adopt a distinct morphology. Since cell spreading is dependent on the integrity of the actin microfilament cytoskeleton, we sought to determine if actin depolymerization was sufficient to induce apoptosis, even in the presence of continuous attachment. For this study, we used a human mammary epithelial cell line (MCF10A), which is immortalized but remains adhesion dependent for survival. Treatment of MCF10A cells with latrunculin-A (LA), an inhibitor of actin polymerization, rapidly led to disruption of the actin cytoskeleton and caused cell rounding but preserved attachment. Initiation of apoptosis in LA-treated MCF10A cells was detected by mitochondrial localization of the Bax apoptotic protein, which was prevented by overexpression of Bcl-2. DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage in LA-treated MCF10A cells indicated progression to the execution phase of apoptosis. The MDA-MB-453 cell line, which was derived from a metastatic human mammary tumor, was resistant to PARP cleavage and loss of viability in response to actin depolymerization. Stable overexpression of Bcl-2 in the untransformed MCF10A cells was able to recapitulate the resistance to apoptosis found in the tumor cell line. We demonstrate that inhibition of actin polymerization is sufficient to stimulate apoptosis in attached MCF10A cells, and we present a novel role for Bcl-2 in cell death induced by direct disruption of the actin cytoskeleton.

  19. Triggering Actin Comets Versus Membrane Ruffles: Distinctive Effects of Phosphoinositides on Actin Reorganization

    PubMed Central

    Ueno, Tasuku; Falkenburger, Björn H.; Pohlmeyer, Christopher; Inoue, Takanari

    2012-01-01

    A limited set of phosphoinositide membrane lipids regulate diverse cellular functions including proliferation, differentiation, and migration. We developed two techniques based on rapamycin-induced protein dimerization to rapidly change the concentration of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. First, we increased PI(4,5)P2 synthesis from phosphatidylinositol 4-phosphate [PI(4)P] using a membrane recruitable form of PI(4)P 5-kinase, and found that COS-7, HeLa, and HEK293 cells formed bundles of motile actin filaments known as actin comets. In contrast, a second technique that increased the concentration of PI(4,5)P2 without consuming PI(4)P induced membrane ruffles. These distinct phenotypes were mediated by dynamin-mediated vesicular trafficking and mutually inhibitory crosstalk between the small guanosine triphosphatases Rac and RhoA. Our results indicate that the effect of PI(4,5)P2 on actin reorganization depends on the abundance of other phosphoinositides, such as PI(4)P. Thus, combinatorial regulation of phosphoinositide concentrations may contribute to the diversity of phosphoinositide functions. PMID:22169478

  20. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  1. Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

    PubMed Central

    Piccinotti, Silvia

    2013-01-01

    Rabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin. PMID:23966407

  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. Actin-cytoskeleton dynamics in non-monotonic cell spreading

    PubMed Central

    Heinrich, Doris; Youssef, Simon; Schroth-Diez, Britta; Engel, Ulrike; Aydin, Daniel; Blümmel, Jacques; Spatz, Joachim P

    2008-01-01

    The spreading of motile cells on a substrate surface is accompanied by reorganization of their actin network. We show that spreading in the highly motile cells of Dictyostelium is non-monotonic, and thus differs from the passage of spreading cells through a regular series of stages. Quantification of the gain and loss of contact area revealed fluctuating forces of protrusion and retraction that dominate the interaction of Dictyostelium cells with a substrate. The molecular basis of these fluctuations is elucidated by dual-fluorescence labeling of filamentous actin together with proteins that highlight specific activities in the actin system. Front-to-tail polarity is established by the sorting out of myosin-II from regions where dense actin assemblies are accumulating. Myosin-IB identifies protruding front regions, and the Arp2/3 complex localizes to lamellipodia protruded from the fronts. Coronin is used as a sensitive indicator of actin disassembly to visualize the delicate balance of polymerization and depolymerization in spreading cells. Short-lived actin patches that co-localize with clathrin suggest that membrane internalization occurs even when the substrate-attached cell surface expands. We conclude that non-monotonic cell spreading is characterized by spatiotemporal patterns formed by motor proteins together with regulatory proteins that either promote or terminate actin polymerization on the scale of seconds. PMID:19262103

  4. Drebrin attenuates the interaction between actin and myosin-V.

    PubMed

    Ishikawa, Ryoki; Katoh, Kaoru; Takahashi, Ayumi; Xie, Ce; Oseki, Koushi; Watanabe, Michitoshi; Igarashi, Michihiro; Nakamura, Akio; Kohama, Kazuhiro

    2007-07-27

    Drebrin-A is an actin-binding protein localized in the dendritic spines of mature neurons, and has been suggested to affect spine morphology [K. Hayashi, T. Shirao, Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons, J. Neurosci. 19 (1999) 3918-3925]. However, no biochemical analysis of drebrin-A has yet been reported. In this study, we purified drebrin-A using a bacterial expression system, and characterized it in vitro. Drebrin-A bound to actin filaments with a stoichiometry of one drebrin molecule to 5-6 actin molecules. Furthermore, drebrin-A decreased the Mg-ATPase activity of myosin V. In vitro motility assay revealed that the attachment of F-actin to glass surface coated with myosin-V was decreased by drebrin-A, but once F-actin attached to the surface, the sliding speed of F-actin was unaffected by the presence of drebrin A. These findings suggest that drebrin-A may affect spine dynamics, vesicle transport, and other myosin-V-driven motility in neurons through attenuating the interaction between actin and myosin-V.

  5. Simulation of the effect of confinement in actin ring formation

    NASA Astrophysics Data System (ADS)

    Adeli Koudehi, Maral; Vavylonis, Dimitrios; Haosu Tang Team; Dimitrios Vavylonis Team

    Actin filaments are vital for different network structures in living cells. During cytokinesis, they form a contractile ring containing myosin motor proteins and actin filament cross-linkers to separate one cell into two cells. Recent experimental studies have quantified the bundle, ring, and network structures that form when actin filaments polymerize in confined environments in vitro, in the presence of varying concentrations of cross-linkers. In this study, we performed numerical simulations to investigate the effect of actin spherical confinement and cross-linking in ring formation. We used a spring-bead model and Brownian dynamics to simulate semiflexible actin filaments that polymerize in a confining sphere with a rate proportional to the monomer concentration. Applying the model for different size of the confining spheres shows that the probability of ring formation decreases by increasing the radius (at fixed initial monomer concentration), in agreement with prior experimental data. We describe the effect of persistence length, orientation-dependent cross-linking, and initial actin monomer concentration. Simulations show that equilibrium configurations can be reached through zipping and unzipping of actin filaments in bundles and transient ring formation.

  6. Quantitative fluorescent speckle microscopy (QFSM) to measure actin dynamics.

    PubMed

    Mendoza, Michelle C; Besson, Sebastien; Danuser, Gaudenz

    2012-10-01

    Quantitative fluorescent speckle microscopy (QFSM) is a live-cell imaging method to analyze the dynamics of macromolecular assemblies with high spatial and temporal resolution. Its greatest successes were in the analysis of actin filament and adhesion dynamics in the context of cell migration and microtubule dynamics in interphase and the meiotic/mitotic spindle. Here, focus is on the former application to illustrate the procedures of FSM imaging and the computational image processing that extracts quantitative information from these experiments. QFSM is advantageous over other methods because it measures the movement and turnover kinetics of the actin filament (F-actin) network in living cells across the entire field of view. Experiments begin with the microinjection of fluorophore-labeled actin into cells, which generate a low ratio of fluorescently labeled to endogenously unlabeled actin monomers. Spinning disk confocal or wide-field imaging then visualizes fluorophore clusters (two to eight actin monomers) within the assembled F-actin network as speckles. QFSM software identifies and computationally tracks and utilizes the location, appearance, and disappearance of speckles to derive network flows and maps of the rate of filament assembly and disassembly. PMID:23042526

  7. Interconnection between actin cytoskeleton and plant defense signaling.

    PubMed

    Janda, Martin; Matoušková, Jindřiška; Burketová, Lenka; Valentová, Olga

    2014-01-01

    Actin cytoskeleton is the fundamental structural component of eukaryotic cells. It has a role in numerous elementary cellular processes such as reproduction, development and also in response to abiotic and biotic stimuli. Remarkably, the role of actin cytoskeleton in plant response to pathogens is getting to be under magnifying glass. Based on microscopic studies, most of the data showed, that actin plays an important role in formation of physiological barrier in the site of infection. Actin dynamics is involved in the transport of antimicrobial compounds and cell wall fortifying components (e.g. callose) to the site of infection. Also the role in PTI (pathogen triggered immunity) and ETI (effector triggered immunity) was recently indicated. On the other hand much less is known about the transcriptome reprogramming upon changes in actin dynamics. Our recently published results showed that drugs inhibiting actin polymerization (latrunculin B, cytochalasin E) cause the induction of genes which are involved in salicylic acid (SA) signaling pathway. In this addendum we would like to highlight in more details current state of knowledge concerning the involvement of actin dynamics in plant defense signaling.

  8. The centrosome is an actin-organizing center

    PubMed Central

    Farina, Francesca; Gaillard, Jérémie; Guérin, Christophe; Couté, Yohann; Sillibourne, James; Blanchoin, Laurent; Théry, Manuel

    2016-01-01

    Microtubules and actin filaments are the two main cytoskeleton networks supporting intracellular architecture and cell polarity. The centrosome nucleates and anchors microtubules and is therefore considered to be the main microtubule-organizing center. However, recurring, yet unexplained, observations have pointed towards a connection between the centrosome and actin filaments. Here we have used isolated centrosomes to demonstrate that the centrosome can directly promote actin filament assembly. A cloud of centrosome-associated actin filaments could be identified in living cells as well. Actin-filament nucleation at the centrosome was mediated by the nucleation promoting factor WASH in combination with the Arp2/3 complex. Pericentriolar material 1 (PCM1) appeared to modulate the centrosomal actin network by regulating Arp2/3 complex and WASH recruitment to the centrosome. Hence our results reveal an additional facet of the centrosome as an intracellular organizer and provide mechanistic insights into how the centrosome can function as an actin filament-organizing center. PMID:26655833

  9. Symmetry breaking in actin gels - Implications for cellular motility

    NASA Astrophysics Data System (ADS)

    John, Karin; Peyla, Philippe; Misbah, Chaouqi

    2007-03-01

    The physical origin of cell motility is not fully understood. Recently minimal model systems have shown, that polymerizing actin itself can produce a motile force, without the help of motor proteins. Pathogens like Shigella or Listeria use actin to propel themselves forward in their host cell. The same process can be mimicked with polystyrene beads covered with the activating protein ActA, which reside in a solution containing actin monomers. ActA induces the growth of an actin gel at the bead surface. Initially the gel grows symmetrically around the bead until a critical size is reached. Subsequently one observes a symmetry breaking and the gel starts to grow asymmetrically around the bead developing a tail of actin at one side. This symmetry breaking is accompanied by a directed movement of the bead, with the actin tail trailing behind the bead. Force generation relies on the combination of two properties: growth and elasticity of the actin gel. We study this phenomenon theoretically within the framework of a linear elasticity theory and linear flux-force relationships for the evolution of an elastic gel around a hard sphere. Conditions for a parity symmetry breaking are identified analytically and illustrated numerically with the help of a phasefield model.

  10. Phosphorylation of platelet actin-binding protein during platelet activation

    SciTech Connect

    Carroll, R.C.; Gerrard, J.M.

    1982-03-01

    In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide-sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2 to 3-fold labeling increases in actin-binding protein 30 to 60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P-labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

  11. Role of actin in auxin transport and transduction of gravity

    NASA Astrophysics Data System (ADS)

    Hu, S.; Basu, S.; Brady, S.; Muday, G.

    Transport of the plant hormone auxin is polar and the direction of the hormone movement appears to be controlled by asymmetric distribution of auxin transport protein complexes. Changes in the direction of auxin transport are believed to drive asymmetric growth in response to changes in the gravity vector. To test the possibility that asymmetric distribution of the auxin transport protein complex is mediated by attachment to the actin cytoskeleton, a variety of experimental approaches have been used. The most direct demonstration of the role of the actin cytoskeleton in localization of the protein complex is the ability of one protein in this complex to bind to affinity columns containing actin filaments. Additionally, treatments of plant tissues with drugs that fragment the actin c toskeleton reducey polar transport. In order to explore this actin interaction and the affect of gravity on auxin transport and developmental polarity, embryos of the brown alga, Fucus have been examined. Fucus zygotes are initially symmetrical, but develop asymmetry in response to environmental gradients, with light gradients being the best- characterized signal. Gravity will polarize these embryos and gravity-induced polarity is randomized by clinorotation. Auxin transport also appears necessary for environmental controls of polarity, since auxin efflux inhibitors perturb both photo- and gravity-polarization at a very discrete temporal window within six hours after fertilization. The actin cytoskeleton has previously been shown to reorganize after fertilization of Fucus embryos leading to formation of an actin patch at the site of polar outgrowth. These actin patches still form in Fucus embryos treated with auxin efflux inhibitors, yet the position of these patches is randomized. Together, these results suggest that there are connections between the actin cytoskeleton, auxin transport, and gravity oriented growth and development. (Supported by NASA Grant: NAG2-1203)

  12. The structure of the cuticular plate, an in vivo actin gel.

    PubMed

    DeRosier, D J; Tilney, L G

    1989-12-01

    The cuticular plate is a network of actin filaments found in hair cells of the cochlea. In the alligator lizard, it consists of rootlets, emanating from the stereocilia, and of cross-connecting actin filaments that anchor these rootlets. In thin sections, this network displays striking patches of 650 +/- 110-A striae. By quantitative analyses of the images, the mystery of the striae can be explained. They are due in part to the rootlets which are sets of flat ribbons of actin filaments. The ribbons in each set are separated by approximately 650 A. Numerous whiskers 30 A in diameter extend from each ribbon's face, interconnecting adjacent ribbons. The nonrootlet filaments, except at the margins of the cell, occur primarily as single filaments. Like the ribbons, they are bristling with whiskers. The patches of striae are explained by ribbons and filaments held at a 650-A separation by the whiskers that project from them. A simple model for regions of bewhiskered filaments is a box crammed full of randomly oriented test-tube brushes. A thin slice through the box will show regions of dark lines or striae due to the wire backbones of the brushes separated from one another by the bristle length. Using the computer instead of test-tube brushes, we have been able to model quantitatively the filament distribution and pattern of striae seen in the cuticular plate of the lizard. The organization of actin filaments we have deduced from our simulations differs from that found in macrophages or in the terminal web of intestinal epithelial cells.

  13. The structure of the cuticular plate, an in vivo actin gel.

    PubMed

    DeRosier, D J; Tilney, L G

    1989-12-01

    The cuticular plate is a network of actin filaments found in hair cells of the cochlea. In the alligator lizard, it consists of rootlets, emanating from the stereocilia, and of cross-connecting actin filaments that anchor these rootlets. In thin sections, this network displays striking patches of 650 +/- 110-A striae. By quantitative analyses of the images, the mystery of the striae can be explained. They are due in part to the rootlets which are sets of flat ribbons of actin filaments. The ribbons in each set are separated by approximately 650 A. Numerous whiskers 30 A in diameter extend from each ribbon's face, interconnecting adjacent ribbons. The nonrootlet filaments, except at the margins of the cell, occur primarily as single filaments. Like the ribbons, they are bristling with whiskers. The patches of striae are explained by ribbons and filaments held at a 650-A separation by the whiskers that project from them. A simple model for regions of bewhiskered filaments is a box crammed full of randomly oriented test-tube brushes. A thin slice through the box will show regions of dark lines or striae due to the wire backbones of the brushes separated from one another by the bristle length. Using the computer instead of test-tube brushes, we have been able to model quantitatively the filament distribution and pattern of striae seen in the cuticular plate of the lizard. The organization of actin filaments we have deduced from our simulations differs from that found in macrophages or in the terminal web of intestinal epithelial cells. PMID:2592408

  14. Cleavage of human and mouse cytoskeletal and sarcomeric proteins by human immunodeficiency virus type 1 protease. Actin, desmin, myosin, and tropomyosin.

    PubMed Central

    Shoeman, R. L.; Sachse, C.; Höner, B.; Mothes, E.; Kaufmann, M.; Traub, P.

    1993-01-01

    HeLa cell actin was cleaved by human immunodeficiency virus type 1 protease when in its soluble, globular form (G-actin). No cleavage of the polymerized, filamentous form of actin (F-actin) was observed when examined by denaturing gel electrophoresis; however, electron microscopy revealed a low level of cleavage of F-actin. Immunoblotting of mouse skeletal and human pectoral muscle myofibrils treated in vitro with human immunodeficiency virus type 1 protease showed that myosin heavy chain, desmin, tropomyosin, and a fraction of the actin were all cleaved. Electron microscopy of these myofibrils demonstrated changes consistent with cleavage of these proteins: Z-lines were rapidly lost, the length of the A bands was shortened, and the thick filaments (myosin filaments) were often laterally frayed such that the structures disintegrated. Nonmuscle myosin heavy chains were also cleaved by this enzyme in vitro. These data demonstrate that this protease can cause alterations in muscle cell ultrastructure in vitro that may be of clinical relevance in infected individuals. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:8424456

  15. Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding.

    PubMed

    Borovikov, Y S; Moraczewska, J; Khoroshev, M I; Strzelecka-Gołaszewska, H

    2000-03-16

    Effects of subtilisin cleavage of actin between residues 47 and 48 on the conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers (ghost fibers). In separate experiments, polarized fluorescence from 1, 5-IAEDANS-labeled S1 bound to non-labeled actin filaments in ghost fibers was measured. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-phalloidin-labeled filaments, orientation and mobility of the C-terminus of actin, filament flexibility, and orientation and mobility of the myosin heads bound to F-actin. The changes in the filament flexibility and orientation of the actin-bound fluorophores produced by S1 binding to actin in the absence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in maintaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be essential for the generation of force and movement during actin-myosin interaction.

  16. Actin purification from a gel of rat brain extracts.

    PubMed

    Levilliers, N; Peron-Renner, M; Coffe, G; Pudles, J

    1984-01-01

    Actin, 99% pure, has been recovered from rat brain with a high yield (greater than 15 mg/100 g brain). We have shown that: 1. a low ionic strength extract from rat brain tissue is capable of giving rise to a gel; 2. actin is the main gel component and its proportion is one order of magnitude higher than in the original extract; 3. actin can be isolated from this extract by a three-step procedure involving gelation, dissociation of the gel in 0.6 M KCl, followed by one or two depolymerization-polymerization cycles. PMID:6529588

  17. Actinic reticuloid imitating Sézary syndrome.

    PubMed

    Pacheco, David; Fraga, Ana; Travassos, Ana Rita; Antunes, Joana; Freitas, Joaao; Soares de Almeida, Luis; Filipe, Paulo

    2012-09-01

    Actinic reticuloid is a rare chronic idiopathic photosensitive dermatosis belonging to the spectrum of chronic actinic dermatitis and may be mistaken for cutaneous T-cell lymphoma. We report the case of an erythrodermic patient, initially diagnosed with Sézary syndrome, treated with chlorambucil and prednisolone. Later on, a photobiological study demonstrated photosensitivity to UVB, UVA, and visible light. The clinical picture, histological findings resembling lymphoma, and the results of the photobiological study established the diagnosis of actinic reticuloid. Currently the patient is avoiding sun exposure and being medicated with azathioprine and prednisolone, and is showing sustained improvement. PMID:23267873

  18. Acanthamoeba castellanii: identification and distribution of actin cytoskeleton.

    PubMed

    González-Robles, Arturo; Castañón, Guadalupe; Hernández-Ramírez, Verónica Ivonne; Salazar-Villatoro, Lizbeth; González-Lázaro, Mónica; Omaña-Molina, Maritza; Talamás-Rohana, Patricia; Martínez-Palomo, Adolfo

    2008-07-01

    The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmicroscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks of actin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endocytic structures. In addition, the comparison between total actin content in whole extracts among different amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa for Naegleria fowleri and Entamoeba histolytica.

  19. Evolution of the actin gene family in testate lobose amoebae (Arcellinida) is characterized by two distinct clades of paralogs and recent independent expansions.

    PubMed

    Lahr, Daniel J G; Nguyen, Truc B; Barbero, Erika; Katz, Laura A

    2011-01-01

    The evolution of actin gene families is characterized by independent expansions and contractions across the eukaryotic tree of life. Here, we assess diversity of actin gene sequences within three lineages of the genus Arcella, a free-living testate (shelled) amoeba in the Arcellinida. We established four clonal lines of two morphospecies, Arcella hemisphaerica and A. vulgaris, and assessed their phylogenetic relationship within the "Amoebozoa" using small subunit ribosomal DNA (SSU-rDNA) genealogy. We determined that the two lines of A. hemisphaerica are identical in SSU-rDNA, while the two A. vulgaris are independent genetic lineages. Furthermore, we characterized multiple actin gene copies from all lineages. Analyses of the resulting sequences reveal numerous diverse actin genes, which differ mostly by synonymous substitutions. We estimate that the actin gene family contains 40-50 paralogous members in each lineage. None of the three independent lineages share the same paralog with another, and divergence between actins reaches 29% in contrast to just 2% in SSU-rDNA. Analyses of effective number of codons (ENC), compositional bias, recombination signatures, and genetic diversity in the context of a gene tree indicate that there are two groups of actins evolving with distinct patterns of molecular evolution. Within these groups, there have been multiple independent expansions of actin genes within each lineage. Together, these data suggest that the two groups are located in different regions of the Arcella genome. Furthermore, we compare the Arcella actin gene family with the relatively well-described gene family in the slime mold Dictyostelium discoideum and other members of the Amoebozoa clade. Overall patterns of molecular evolution are similar in Arcella and Dictyostelium. However, the separation of genes in two distinct groups coupled with recent expansion is characteristic of Arcella and might reflect an unusual pattern of gene family evolution in the lobose

  20. Evolution of the Actin Gene Family in Testate Lobose Amoebae (Arcellinida) is Characterized by Two Distinct Clades of Paralogs and Recent Independent Expansions

    PubMed Central

    Lahr, Daniel J. G.; Nguyen, Truc B.; Barbero, Erika; Katz, Laura A.

    2011-01-01

    The evolution of actin gene families is characterized by independent expansions and contractions across the eukaryotic tree of life. Here, we assess diversity of actin gene sequences within three lineages of the genus Arcella, a free-living testate (shelled) amoeba in the Arcellinida. We established four clonal lines of two morphospecies, Arcella hemisphaerica and A. vulgaris, and assessed their phylogenetic relationship within the “Amoebozoa” using small subunit ribosomal DNA (SSU-rDNA) genealogy. We determined that the two lines of A. hemisphaerica are identical in SSU-rDNA, while the two A. vulgaris are independent genetic lineages. Furthermore, we characterized multiple actin gene copies from all lineages. Analyses of the resulting sequences reveal numerous diverse actin genes, which differ mostly by synonymous substitutions. We estimate that the actin gene family contains 40–50 paralogous members in each lineage. None of the three independent lineages share the same paralog with another, and divergence between actins reaches 29% in contrast to just 2% in SSU-rDNA. Analyses of effective number of codons (ENC), compositional bias, recombination signatures, and genetic diversity in the context of a gene tree indicate that there are two groups of actins evolving with distinct patterns of molecular evolution. Within these groups, there have been multiple independent expansions of actin genes within each lineage. Together, these data suggest that the two groups are located in different regions of the Arcella genome. Furthermore, we compare the Arcella actin gene family with the relatively well-described gene family in the slime mold Dictyostelium discoideum and other members of the Amoebozoa clade. Overall patterns of molecular evolution are similar in Arcella and Dictyostelium. However, the separation of genes in two distinct groups coupled with recent expansion is characteristic of Arcella and might reflect an unusual pattern of gene family evolution in the

  1. Curved trajectories of actin-based motility in two dimensions

    NASA Astrophysics Data System (ADS)

    Wen, Fu-Lai; Leung, Kwan-tai; Chen, Hsuan-Yi

    2012-05-01

    Recent experiments have reported fascinating geometrical trajectories for actin-based motility of bacteria Listeria monocytogenes and functionalized beads. To understand the physical mechanism for these trajectories, we constructed a phenomenological model to study the motion of an actin-propelled disk in two dimensions. In our model, the force and actin density on the surface of the disk are influenced by the translation and rotation of the disk, which in turn is induced by the asymmetric distributions of those densities. We show that this feedback can destabilize a straight trajectory, leading to circular, S-shape and other geometrical trajectories observed in the experiments through bifurcations in the distributions of the force and actin density. The relation between our model and the models for self-propelled deformable particles is emphasized and discussed.

  2. Tracing myoblast fusion in Drosophila embryos by fluorescent actin probes.

    PubMed

    Haralalka, Shruti; Abmayr, Susan M

    2015-01-01

    Myoblast fusion in the Drosophila embryo is a highly elaborate process that is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs). It occurs through an asymmetric event in which actin foci assemble in the FCMs at points of cell-cell contact and direct the formation of membrane protrusions that drive fusion. Herein, we describe the approach that we have used to image in living embryos the highly dynamic actin foci and actin-rich projections that precede myoblast fusion. We discuss resources currently available for imaging actin and myogenesis, and our experience with these resources if available. This technical report is not intended to be comprehensive on providing instruction on standard microscopy practices or software utilization. However, we discuss microscope parameters that we have used in data collection, and our experience with image processing tools in data analysis.

  3. Cortactin Branches Out: Roles in Regulating Protrusive Actin Dynamics

    PubMed Central

    Ammer, Amanda Gatesman; Weed, Scott A.

    2008-01-01

    Since its discovery in the early 1990’s, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures. PMID:18615630

  4. Measuring actin dynamics during phagocytosis using photo-switchable fluorescence

    NASA Astrophysics Data System (ADS)

    Kovari, Daniel T.; Curtis, Jennifer E.

    2013-03-01

    Phagocytosis has traditionally been investigated in terms of the relevant biochemical signaling pathways. However, a growing number of studies investigating the physical aspects of phagocytosis have demonstrated that several distinct forces are exerted throughout particle ingestion. We use variations on FRAP (Fluorescence Recovery After Photobleaching) in combination with photo-switchable fluorescent protein to investigate actin dynamics as a phagocyte attempts to engulf its prey. The goal of our actin studies are to determine the recruitment and polymerization rate of actin in the forming phagosome and whether an organized contractile actin ring is present and responsible for phagosome closure, as proposed in the literature. These experiments are ongoing and contribute to our long term effort of developing a physics based model of phagocytosis.

  5. Induction of anti-actin drug resistance in Tetrahymena.

    PubMed

    Zackroff, Robert V; Hufnagel, Linda A

    2002-01-01

    Both cytochalasin D and latrunculin B reversibly inhibited Tetrahymena phagocytosis at concentrations similar to those effective in mammalian systems, even though ciliate actins are known to be highly divergent from mammalian actins. Overnight exposure to relatively low (0.25 microM) concentrations of latrunculin B induced resistance in Tetrahymena to the inhibitory effects of that drug, as well as cross-resistance to cytochalasin D. However, much higher (> 30 microM) concentrations of cytochalasin D were required for induction of cross-resistance to latrunculin B. Anti-actin drug resistance in Tetrahymena may involve a general multidrug resistance mechanism and/or specific feedback regulation of F-actin assembly and stability.

  6. Stable high capacity, F-actin affinity column

    SciTech Connect

    Luna, E.J.; Wang, Y.L.; Voss, E.W. Jr.; Branton, D.; Taylor, D.L.

    1982-11-10

    A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds /sup 125/I-heavy meromyosin and /sup 125/I-tropomyosin. The associations between the F-actin-binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.

  7. A model actin comet tail disassembling by severing

    PubMed Central

    Michalski, P J; Carlsson, A E

    2011-01-01

    We use a numerical simulation to model an actin comet tail as it grows from the surface of a small object (a bead) and disassembles by severing. We explore the dependence of macroscopic properties such as the local tail radius and tail length on several controllable properties, namely, the bead diameter, the bead velocity, the severing rate per unit length, and the actin gel mesh size. The model predicts an F-actin density with an initial exponential decay followed by an abrupt decay at the edge of the tail, and predicts that the comet tail diameter is constant along the length of the tail. The simulation results are used to fit a formula relating the comet tail length to the control parameters, and it is proposed that this formula offers a means to extract quantitative information on the actin gel mesh size and severing kinetics from simple macroscopic measurements. PMID:21566272

  8. The role of actin networks in cellular mechanosensing

    NASA Astrophysics Data System (ADS)

    Azatov, Mikheil

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

  9. Upstream regions of the human cardiac actin gene that modulate its transcription in muscle cells: presence of an evolutionarily conserved repeated motif.

    PubMed Central

    Minty, A; Kedes, L

    1986-01-01

    Transfection into cultured cell lines was used to investigate the transcriptional regulation of the human cardiac actin gene. We first demonstrated that in both human heart and human skeletal muscle, cardiac actin mRNAs initiate at the identical site and contain the same first exon, which is separated from the first coding exon by an intron of 700 base pairs. A region of 485 base pairs upstream from the transcription initiation site of the human cardiac actin gene directs high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated myotubes of the mouse C2C12 muscle cell line, but not in mouse L fibroblast or rat PC-G2 pheochromocytoma cells. Deletion analysis of this region showed that at least two physically separated sequence elements are involved, a distal one starting between -443 and -395 and a proximal one starting between -177 and -118, and suggested that these sequences interact with positively acting transcriptional factors in muscle cells. When these two sequence elements are inserted separately upstream of a heterologous (simian virus 40) promoter, they do not affect transcription but do give a small (four- to fivefold) stimulation when tested together. Overall, these regulatory regions upstream of the cap site of the human cardiac actin gene show remarkably high sequence conservation with the equivalent regions of the mouse and chick genes. Furthermore, there is an evolutionarily conserved repeated motif that may be important in the transcriptional regulation of actin and other contractile protein genes. Images PMID:3785189

  10. The core and conserved role of MAL is homeostatic regulation of actin levels.

    PubMed

    Salvany, Lara; Muller, Julius; Guccione, Ernesto; Rørth, Pernille

    2014-05-15

    The transcription cofactor MAL is regulated by free actin levels and thus by actin dynamics. MAL, together with its DNA-binding partner, SRF, is required for invasive cell migration and in experimental metastasis. Although MAL/SRF has many targets, we provide genetic evidence in both Drosophila and human cellular models that actin is the key target that must be regulated by MAL/SRF for invasive cell migration. By regulating MAL/SRF activity, actin protein feeds back on production of actin mRNA to ensure sufficient supply of actin. This constitutes a dedicated homeostatic feedback system that provides a foundation for cellular actin dynamics.

  11. Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation

    PubMed Central

    1988-01-01

    The expression of cytoplasmic beta-actin and cardiac, skeletal, and smooth muscle alpha-actins during early avian cardiogenesis was analyzed by in situ hybridization with mRNA-specific single-stranded DNA probes. The cytoplasmic beta-actin gene was ubiquitously expressed in the early chicken embryo. In contrast, the alpha-actin genes were sequentially activated in avian cardiac tissue during the early stages of heart tube formation. The accumulation of large quantities of smooth muscle alpha-actin transcripts in epimyocardial cells preceded the expression of the sarcomeric alpha-actin genes. The accumulation of skeletal alpha-actin mRNAs in the developing heart lagged behind that of cardiac alpha-actin by several embryonic stages. At Hamburger- Hamilton stage 12, the smooth muscle alpha-actin gene was selectively down-regulated in the heart such that only the conus, which subsequently participates in the formation of the vascular trunks, continued to express this gene. This modulation in smooth muscle alpha- actin gene expression correlated with the beginning of coexpression of sarcomeric alpha-actin transcripts in the epimyocardium and the onset of circulation in the embryo. The specific expression of the vascular smooth muscle alpha-actin gene marks the onset of differentiation of cardiac cells and represents the first demonstration of coexpression of both smooth muscle and striated alpha-actin genes within myogenic cells. PMID:3204121

  12. Comparison of actin and cell surface dynamics in motile fibroblasts

    PubMed Central

    1992-01-01

    We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton. PMID:1400580

  13. Positioning and stretching of actin filaments by electric fields

    NASA Astrophysics Data System (ADS)

    Wigge, Christoph; Hinssen, Horst; Reiss, Günter; Herth, Simone

    2010-06-01

    The alignment of biological filaments on surfaces offers a high potential for controllable geometries in lab-on-a-chip-structures and micrototal analysis systems. Actin is a polar filamentous protein with a diameter of 7-8 nm that can be manipulated with strong electric fields. It is demonstrated that with the use of microelectrodes or nanoelectrodes and electric fields of 20 kV/m single actin filaments can be manipulated, stretched, and positioned between gold electrodes.

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

    SciTech Connect

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

    2013-11-29

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

  15. Both actin and polyproline interactions of Profilin-1 are required for migration, invasion and capillary morphogenesis of vascular endothelial cells

    PubMed Central

    Ding, Zhijie; Gau, David; Deasy, Bridget; Wells, Alan; Roy, Partha

    2009-01-01

    The objective of the present study was to evaluate how different ligand interactions of profilin-1 (Pfn1), an actin-binding protein that is upregulated during capillary morphogenesis of vascular endothelial cells (VEC), contribute to migration and capillary forming ability of VEC. We adopted a knockdown-knockin experimental system to stably express either fully-functional or mutants of Pfn1 that are impaired in binding to two of its major ligands, actin (H119E mutant) and proteins containing polyproline domains (H133S mutant), in a human dermal microvascular cell line (HmVEC) against near-null endogenous Pfn1 background. We found that silencing endogenous Pfn1 expression in HmVEC leads to slower random migration, reduced velocity of membrane protrusion and a significant impairment in matrigel-induced cord formation. Only re-expression of fully-functional but not any of the two ligand-binding deficient mutants of Pfn1 rescues the above defects. We further show that loss of Pfn1 expression in VEC inhibits three-dimensional capillary morphogenesis, MMP2 secretion and ECM invasion. VEC invasion through ECM is also inhibited when actin and polyproline interactions of Pfn1 are disrupted. Together, these experimental data demonstrate that Pfn1 regulates VEC migration, invasion and capillary morphogenesis through its interaction with both actin and proline-rich ligands. PMID:19607826

  16. Fucus as a Model System to Study the Role of Auxin Transport and the Actin Cytoskeleton in Gravity Response

    NASA Technical Reports Server (NTRS)

    Muday, Gloria K.

    2003-01-01

    The overarching goal of this proposal was to examine the mechanisms for the cellular asymmetry in auxin transport proteins. As auxin transport polarity changes in response to reorientation of algal and plant cells relative to the gravity vector, it was critical to ask how auxin transport polarity is established and how this transport polarity may change in response to gravity stimulation. The experiments conducted with this NASA grant fell into two categories. The first area of experimentation was to explore the biochemical interactions between an auxin transport protein and the actin cytoskeleton. These experiments used biochemical techniques, including actin affinity chromatography, to demonstrate that one auxin transport protein interacts with the actin cytoskeleton. The second line of experiments examined whether in the initially symmetrical single celled embryos of Fucus distichus, whether auxin regulates development and whether gravity is a cue to control the morphogenesis of these embryos and whether gravi-morphogenesis is auxin dependent. Results in these two areas are summarized separately below. As a result of this funding, in combination with results from other investigators, we have strong evidence for an important role for the actin cytoskeleton in both establishing and change auxin transport polarity. It is also clear that Fucus distichus embryos are auxin responsive and gravity controls their morphogenesis.

  17. In Vivo Imaging and Characterization of Actin Microridges

    PubMed Central

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

    2015-01-01

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

  18. Regulation of Actin by Ion-Linked Equilibria

    PubMed Central

    Kang, Hyeran; Bradley, Michael J.; Elam, W. Austin; De La Cruz, Enrique M.

    2013-01-01

    Actin assembly, filament mechanical properties, and interactions with regulatory proteins depend on the types and concentrations of salts in solution. Salts modulate actin through both nonspecific electrostatic effects and specific binding to discrete sites. Multiple cation-binding site classes spanning a broad range of affinities (nanomolar to millimolar) have been identified on actin monomers and filaments. This review focuses on discrete, low-affinity cation-binding interactions that drive polymerization, regulate filament-bending mechanics, and modulate interactions with regulatory proteins. Cation binding may be perturbed by actin post-translational modifications and linked equilibria. Partial cation occupancy under physiological and commonly used in vitro solution conditions likely contribute to filament mechanical heterogeneity and structural polymorphism. Site-specific cation-binding residues are conserved in Arp2 and Arp3, and may play a role in Arp2/3 complex activation and actin-filament branching activity. Actin-salt interactions demonstrate the relevance of ion-linked equilibria in the operation and regulation of complex biological systems. PMID:24359734

  19. Myelin basic protein domains involved in the interaction with actin.

    PubMed

    Roth, G A; Gonzalez, M D; Monferran, C G; De Santis, M L; Cumar, F A

    1993-11-01

    A fluorescence assay was used to measure the interaction of myelin basic protein (MBP) with monomeric actin labeled with a fluorescent compound (IAEDANS). The complex actin-IAEDANS increase the fluorescence in presence of MBP. The enhancement of the fluorescence has a sigmoidal dependence on the concentration of MBP and the fluorescence maximum is reached at a MBP:actin molar ratio of 1:20. The fluorescence maximum in absence of Ca2+ and ATP is 4 times lower than that in their presence although it is reached at the same MBP:actin molar ratio. Similar behavior is observed when synapsin replaces MBP, while acetylated MBP and bovine serum albumin fail to induce any fluorescence change. To define possible interacting domains on MBP involved in the actin-MBP interaction, experiments were performed using MBP-derived peptides obtained under controlled proteolysis of the whole molecule. The fluorescence changes induced by the different peptides depend on their location in the native protein and can not be explained simply by a difference in the net charge of the peptides. The results suggest that two sites are involved in the interaction. A Ca2+/ATP-dependent site located in the amino-terminal region (peptide 1-44) and a Ca2+/ATP-independent one near the carboxyl terminus of the MBP molecule. The actin-MBP interaction was also observed using immunoblot and ELISA techniques.

  20. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors.

    PubMed

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

    2015-01-01

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

  1. Microstructure and Mechanical Properties of Composite Actin Networks

    NASA Astrophysics Data System (ADS)

    Gardel, Margaret; Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul; Weitz, D. A.

    2003-03-01

    There exits a family of actin-binding proteins (ABPs) and each protein has a distinct function for bundling, networking, gelating, capping, or simply binding to actin. Whether actin serves as a structural or motile component, its mechanical properties are determined by its degree and kinds of association with different ABPs and these properties are often closely related to its functional needs. For instance, in a cell actin is highly crosslinked with multiple ABPs (fimbrin, alpha-actinin, etc.) to generate thrust and strength for locomotion. In the acrosomal reaction of horseshoe crab sperm, actin exists as a bundle of preassembled filaments crosslinked with scruin to form a rigid structure to penetrate into an egg without yielding. We study the effects three different ABPs (scruin,fimbrin and alpha-actinin) have on the rheology and microstructure of actin networks using multiparticle tracking, imaging, and bulk rheology. From these experiments we can deduce how an evolving microstructure affects the bulk rheological properties and the role different concentrations and kinds of ABPs have in these changes.

  2. Actin gene family dynamics in cryptomonads and red algae.

    PubMed

    Tanifuji, Goro; Archibald, John M

    2010-09-01

    Here we present evidence for a complex evolutionary history of actin genes in red algae and cryptomonads, a group that acquired photosynthesis secondarily through the engulfment of a red algal endosymbiont. Four actin genes were found in the nuclear genome of the cryptomonad, Guillardia theta, and in the genome of the red alga, Galdieria sulphuraria, a member of the Cyanidiophytina. Phylogenetic analyses reveal that the both organisms possess two distinct sequence types, designated "type-1" and "type-2." A weak but consistent phylogenetic affinity between the cryptomonad type-2 sequences and the type-2 sequences of G. sulphuraria and red algae belonging to the Rhodophytina was observed. This is consistent with the possibility that the cryptomonad type-2 sequences are derived from the red algal endosymbiont that gave rise to the cryptomonad nucleomorph and plastid. Red algae as a whole possess two very different actin sequence types, with G. sulphuraria being the only organism thus far known to possess both. The common ancestor of Rhodophytina and Cyanidiophytina may have had two actin genes, with differential loss explaining the distribution of these genes in modern-day groups. Our study provides new insight into the evolution and divergence of actin genes in cryptomonads and red algae, and in doing so underscores the challenges associated with heterogeneity in actin sequence evolution and ortholog/paralog detection.

  3. Single-Molecule Studies of Actin Assembly and Disassembly Factors

    PubMed Central

    Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

    2014-01-01

    The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

  4. Actin kinetics shapes cortical network structure and mechanics

    PubMed Central

    Fritzsche, Marco; Erlenkämper, Christoph; Moeendarbary, Emad; Charras, Guillaume; Kruse, Karsten

    2016-01-01

    The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs. PMID:27152338

  5. Single Filaments to Reveal the Multiple Flavors of Actin.

    PubMed

    Jégou, Antoine; Romet-Lemonne, Guillaume

    2016-05-24

    A number of key cell processes rely on specific assemblies of actin filaments, which are all constructed from nearly identical building blocks: the abundant and extremely conserved actin protein. A central question in the field is to understand how different filament networks can coexist and be regulated. Discoveries in science are often related to technical advances. Here, we focus on the ongoing single filament revolution and discuss how these techniques have greatly contributed to our understanding of actin assembly. In particular, we highlight how they have refined our understanding of the many protein-based regulatory mechanisms that modulate actin assembly. It is now becoming apparent that other factors give filaments a specific identity that determines which proteins will bind to them. We argue that single filament techniques will play an essential role in the coming years as we try to understand the many ways actin filaments can take different flavors and unveil how these flavors modulate the action of regulatory proteins. We discuss different factors known to make actin filaments distinguishable by regulatory proteins and speculate on their possible consequences.

  6. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors

    PubMed Central

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

    2015-01-01

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

  7. Cloning and characterization of an actin gene of Chlamys farreri and the phylogenetic analysis of mollusk actins

    NASA Astrophysics Data System (ADS)

    Ma, Hongming; Mai, Kangsen; Liufu, Zhiguo; Xu, Wei

    2007-07-01

    An actingene (CfACTI) was cloned by using RT-PCR, 3’ and 5’RACE from hemocytes of the sea scallop Chlamys farreri. The full length of the transcript is 1 535 bp, which contains a long 3’ un-translated region of 436bp and 59bp of a 5’ un-translated sequence. The open reading frame encodes a polypeptide of 376 amino acids. Sequence comparisons indicated that CfACTI is more closely related to vertebrate cytoplasmic actins than muscle types. Phylogenetic analysis showed that molluscan actins could be generally divided into two categories: muscle and cytoplasmic, although both are similar to vertebrate cytoplasmic actins. It was also inferred that different isotypes existed in muscle or cytoplasma in mollusks. The genomic sequence of CfACTI was cloned and sequenced. Only one intron was detected: it was located between codons 42 and 43 and different from vertebrate actin genes.

  8. Competition for actin between two distinct F-actin networks defines a bistable switch for cell polarization.

    PubMed

    Lomakin, Alexis J; Lee, Kun-Chun; Han, Sangyoon J; Bui, Duyen A; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-11-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype after relaxation of the actomyosin cytoskeleton. We find that myosin II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. Under low-contractility regimes, epithelial cells polarize in a front-back manner owing to the emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin II from the front to the back of the cell, where the motor locally 'locks' actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high-contractility-driven cell motion is inefficient.

  9. Z-line formins promote contractile lattice growth and maintenance in striated muscles of C. elegans

    PubMed Central

    Mi-Mi, Lei; Votra, SarahBeth; Kemphues, Kenneth; Bretscher, Anthony

    2012-01-01

    Muscle contraction depends on interactions between actin and myosin filaments organized into sarcomeres, but the mechanism by which actin filaments incorporate into sarcomeres remains unclear. We have found that, during larval development in Caenorhabditis elegans, two members of the actin-assembling formin family, CYK-1 and FHOD-1, are present in striated body wall muscles near or on sarcomere Z lines, where barbed ends of actin filaments are anchored. Depletion of either formin during this period stunted growth of the striated contractile lattice, whereas their simultaneous reduction profoundly diminished lattice size and number of striations per muscle cell. CYK-1 persisted at Z lines in adulthood, and its near complete depletion from adults triggered phenotypes ranging from partial loss of Z line–associated filamentous actin to collapse of the contractile lattice. These results are, to our knowledge, the first genetic evidence implicating sarcomere-associated formins in the in vivo organization of the muscle cytoskeleton. PMID:22753896

  10. Dynamic Localization of G-actin During Membrane Protrusion in Neuronal Motility

    PubMed Central

    Lee, Chi Wai; Vitriol, Eric A.; Shim, Sangwoo; Wise, Ariel L.; Velayutham, Radhi P.; Zheng, James Q.

    2013-01-01

    Summary Background Actin-based cell motility is fundamental for the development, function, and malignant events of eukaryotic organisms. During neural development, axonal growth cones depend on rapid assembly and disassembly of actin filaments (F-actin) for their guided extension to specific targets for wiring. Monomeric globular actin (G-actin) is the building block for F-actin but is not considered to play a direct role in spatiotemporal control of actin dynamics in cell motility. Results Here we report that a pool of G-actin dynamically localizes to the leading edge of growth cones and neuroblastoma cells to spatially elevate the G-/F-actin ratio that drives membrane protrusion and cell movement. Loss of G-actin localization leads to the cessation and retraction of membrane protrusions. Moreover, G-actin localization occurs asymmetrically in growth cones during attractive turning. Finally, we identify the actin monomer binding proteins profilin and thymosin β4 as key molecules that localize actin monomers to the leading edge of lamellipodia for their motility. Conclusions Our results suggest that dynamic localization of G-actin provides a novel mechanism to regulate the spatiotemporal actin dynamics underlying membrane protrusion in cell locomotion and growth cone chemotaxis. PMID:23746641

  11. F-actin dismantling through a redox-driven synergy between Mical and cofilin.

    PubMed

    Grintsevich, Elena E; Yesilyurt, Hunkar Gizem; Rich, Shannon K; Hung, Ruei-Jiun; Terman, Jonathan R; Reisle