scholarly journals Polymerization of actin by positively charged liposomes.

1988 ◽  
Vol 106 (4) ◽  
pp. 1221-1227 ◽  
Author(s):  
A Laliberte ◽  
C Gicquaud
Keyword(s):  
Electron Microscopy ◽  
Direct Contact ◽  
Atp Hydrolysis ◽  
Actin Assembly ◽  
Liposome Membrane ◽  
Low Salt ◽  
Spectrofluorimetric Analysis ◽  
Positively Charged

By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N-pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts.

scholarly journals Nucleation of polar actin filament assembly by a positively charged surface.

1979 ◽  
Vol 80 (2) ◽  
pp. 499-504 ◽  
Author(s):  
S S Brown ◽  
J A Spudich
Keyword(s):  
Electron Microscopy ◽  
Actin Filament ◽  
Actin Filaments ◽  
Charged Surface ◽  
Actin Assembly ◽  
Filamentous Form ◽  
Filament Assembly ◽  
Biochemical Assays ◽  
Positively Charged

Polylysine-coated polystyrene beads can nucleate polar assembly of monomeric actin into filamentous form. This nucleation has been demonstrated by a combination of biochemical and structural experiments. The polylysine-coated beads accelerate the rate of actin assembly as detected by two different biochemical assays. Subsequent examination of the beads by electron microscopy reveals numerous actin filaments of similar length radiating from the beads. ATP promotes this bead-induced acceleration of assembly. Decoration of the filaments with the myosin fragment S1 shows that these filaments all have the same polarity, with the arrowhead pattern pointing toward the bead. The relevance of the system to in vitro mechanisms and its usefulness in other studies are discussed.

Electron microscopy of a dna helix destabilizing protein crystal (gp32*i)

1978 ◽  
Vol 36 (2) ◽  
pp. 178-179 ◽  
Author(s):  
Wah Chiu ◽  
Junko Hosoda
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Dna Replication ◽  
Bacteriophage T4 ◽  
Polystyrene Latex ◽  
Protein Crystal ◽  
Low Salt ◽  
Leading Strand ◽  
Gene 32

Gp 32 is a protein coded by gene 32 in bacteriophage T4. It is essential for DNA replication, recombination and repair. Gp32*I with molecular weight of 27,000 was obtained by proteolytic removal of 8000-dalton peptide from COOH-terminal of gp32 (Hosoda et al. to be published). It is a stronger helix destabilizer than gp32, and has been found to be able to replace gp32 in constructing an in vitro DNA replication apparatus, which is active in the leading strand synthesis.Thin gp32*I crystal was formed under a low salt condition in the absence of glycerol. Figure 1 shows a typical image of the crystal embedded in 1% glucose taken at low magnification. The optical diffractogram in the insert confirms the crystallinity, though no visible feature is seen in the micrograph due to the low magnification power. By using the tungsten shadowing technique, the thickness of the crystal was measured, along with polystyrene latex spheres as a standard, to be between 85 to 400 Å.

scholarly journals Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

2017 ◽  
Vol 114 (29) ◽  
pp. E5815-E5824 ◽  
Author(s):  
Stefan Brühmann ◽  
Dmitry S. Ushakov ◽  
Moritz Winterhoff ◽  
Richard B. Dickinson ◽  
Ute Curth ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Actin Binding ◽  
Actin Assembly ◽  
Single Filament ◽  
Capping Protein ◽  
Filament Assembly ◽  
Show Evidence ◽  
Membrane Protrusions ◽  
Polypeptide Chains

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin–binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASP-mediated actin assembly in clustered arrays.

scholarly journals Integrated control of formin-mediated actin assembly by a stationary inhibitor and a mobile activator

2018 ◽  
Vol 217 (10) ◽  
pp. 3512-3530 ◽  
Author(s):  
Mikael V. Garabedian ◽  
Tatiana Stanishneva-Konovalova ◽  
Chenyu Lou ◽  
Thomas J. Rands ◽  
Luther W. Pollard ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Integrated Control ◽  
Secretory Vesicles ◽  
Actin Assembly ◽  
Bar Domain ◽  
Binding Partners ◽  
Bud Neck ◽  
Actin Cables

Formins are essential actin assembly factors whose activities are controlled by a diverse array of binding partners. Until now, most formin ligands have been studied on an individual basis, leaving open the question of how multiple inputs are integrated to regulate formins in vivo. Here, we show that the F-BAR domain of Saccharomyces cerevisiae Hof1 interacts with the FH2 domain of the formin Bnr1 and blocks actin nucleation. Electron microscopy of the Hof1–Bnr1 complex reveals a novel dumbbell-shaped structure, with the tips of the F-BAR holding two FH2 dimers apart. Deletion of Hof1’s F-BAR domain in vivo results in disorganized actin cables and secretory defects. The formin-binding protein Bud6 strongly alleviates Hof1 inhibition in vitro, and bud6Δ suppresses hof1Δ defects in vivo. Whereas Hof1 stably resides at the bud neck, we show that Bud6 is delivered to the neck on secretory vesicles. We propose that Hof1 and Bud6 functions are intertwined as a stationary inhibitor and a mobile activator, respectively.

scholarly journals Nucleosome conformational variability in solution and in interphase nuclei evidenced by cryo-electron miocroscopy of vitreous sections

2018 ◽  
Author(s):  
Mikhail Eltsov ◽  
Diana Grewe ◽  
Nicolas Lemercier ◽  
Achilleas Frangakis ◽  
Françoise Livolant ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Interphase Nuclei ◽  
Conformational Variability ◽  
Cryo Electron Microscopy ◽  
Low Salt ◽  
Cellular Context ◽  
The Relationship ◽  
Histone Core

AbstractIn Eukaryotes, DNA is wound around the histone core octamer to form the basic chromatin unit, the nucleosome. Atomic resolution structures have been obtained from crystallography and single particle cryo-electron microscopy of identical engineered particles. But native nucleosomes are dynamical entities with diverse DNA sequence and histone content, and little is known about their conformational variability, especially in the cellular context. Using cryo-electron microscopy and tomography of vitreous sections we analyse the conformation of native nucleosomes, both in vitro, using purified particles solubilised at physiologically relevant concentrations (25-50 %), and in situ, within interphase nuclei. We visualise individual nucleosomes at a level of detail that allows us to analyse the conformation of the DNA wrapped around, and measure the distance between the DNA gyres. We evidence a variety of conformations. In interphase nuclei open nucleosomes predominate, with an average inter-gyre distance larger than that of the canonical particle. In concentrated solutions, we evidence a salt–dependant transition, with high salt compact conformations resembling the canonical nucleosome, and open low salt ones, closer to nuclear nucleosomes. Although further particle characterisation and cartography are needed to understand the relationship between this conformational variability and chromatin functional states, this work opens a route to chromatin exploration in situ.

Sem Studies of Co-Cr-Mo Surgical Implant Alloy Corrosion Behavior

1982 ◽  
Vol 40 ◽  
pp. 520-521
Author(s):  
Ann Chidester Van Orden ◽  
John L. Chidester ◽  
Anna C. Fraker ◽  
Pei Sung
Keyword(s):  
Electron Microscopy ◽  
Corrosion Behavior ◽  
Anodic Polarization ◽  
Saline Solution ◽  
Saturated Calomel Electrode ◽  
Physiological Saline ◽  
X Ray ◽  
Physiological Saline Solution ◽  
Scanning Electron

The influence of small variations in the composition on the corrosion behavior of Co-Cr-Mo alloys has been studied using scanning electron microscopy (SEM), energy dispersive x-ray analysis (EDX), and electrochemical measurements. SEM and EDX data were correlated with data from in vitro corrosion measurements involving repassivation and also potentiostatic anodic polarization measurements. Specimens studied included the four alloys shown in Table 1. Corrosion tests were conducted in Hanks' physiological saline solution which has a pH of 7.4 and was held at a temperature of 37°C. Specimens were mechanically polished to a surface finish with 0.05 µm A1203, then exposed to the solution and anodically polarized at a rate of 0.006 v/min. All voltages were measured vs. the saturated calomel electrode (s.c.e.).. Specimens had breakdown potentials near 0.47V vs. s.c.e.

Maytansine-Induced Mitotic Arrest in Human Lymphoblasts

1977 ◽  
Vol 35 ◽  
pp. 460-461
Author(s):  
Tai-Te Chao ◽  
John Sullivan ◽  
Awtar Krishan
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Transmission Electron Microscopy ◽  
Tubulin Polymerization ◽  
Vinca Alkaloids ◽  
Antimitotic Activity ◽  
Transmission Electron ◽  
Flow Microfluorometry ◽  
Brain Tubulin

Maytansine, a novel ansa macrolide (1), has potent anti-tumor and antimitotic activity (2, 3). It blocks cell cycle traverse in mitosis with resultant accumulation of metaphase cells (4). Inhibition of brain tubulin polymerization in vitro by maytansine has also been reported (3). The C-mitotic effect of this drug is similar to that of the well known Vinca- alkaloids, vinblastine and vincristine. This study was carried out to examine the effects of maytansine on the cell cycle traverse and the fine struc- I ture of human lymphoblasts.Log-phase cultures of CCRF-CEM human lymphoblasts were exposed to maytansine concentrations from 10-6 M to 10-10 M for 18 hrs. Aliquots of cells were removed for cell cycle analysis by flow microfluorometry (FMF) (5) and also processed for transmission electron microscopy (TEM). FMF analysis of cells treated with 10-8 M maytansine showed a reduction in the number of G1 cells and a corresponding build-up of cells with G2/M DNA content.

Some Structural Features of Metaphase Chromosomes of Mice and Men

1967 ◽  
Vol 25 ◽  
pp. 190-191
Author(s):  
Godfrey C. Hoskins ◽  
Betty B. Hoskins
Keyword(s):  
Electron Microscopy ◽  
Electron Micrographs ◽  
Structural Features ◽  
Metaphase Chromosomes ◽  
The Future ◽  
Of Mice And Men ◽  
Mouse Cells ◽  
Human And Mouse

Metaphase chromosomes from human and mouse cells in vitro are isolated by micrurgy, fixed, and placed on grids for electron microscopy. Interpretations of electron micrographs by current methods indicate the following structural features.Chromosomal spindle fibrils about 200Å thick form fascicles about 600Å thick, wrapped by dense spiraling fibrils (DSF) less than 100Å thick as they near the kinomere. Such a fascicle joins the future daughter kinomere of each metaphase chromatid with those of adjacent non-homologous chromatids to either side. Thus, four fascicles (SF, 1-4) attach to each metaphase kinomere (K). It is thought that fascicles extend from the kinomere poleward, fray out to let chromosomal fibrils act as traction fibrils against polar fibrils, then regroup to join the adjacent kinomere.

Electron Microscopy of Fibroblasts from Myotonic Muscular Dystrophy Patients

1981 ◽  
Vol 39 ◽  
pp. 498-499
Author(s):  
S. E. Miller ◽  
G. B. Hartwig ◽  
R. A. Nielsen ◽  
A. P. Frost ◽  
A. D. Roses
Keyword(s):  
Electron Microscopy ◽  
Muscular Dystrophy ◽  
Genetic Diseases ◽  
Skin Fibroblasts ◽  
Inborn Errors ◽  
Cytoplasmic Inclusions ◽  
Cultured Skin ◽  
Myotonic Muscular Dystrophy ◽  
Glycosaminoglycan Metabolism

Many genetic diseases can be demonstrated in skin cells cultured in vitro from patients with inborn errors of metabolism. Since myotonic muscular dystrophy (MMD) affects many organs other than muscle, it seems likely that this defect also might be expressed in fibroblasts. Detection of an alteration in cultured skin fibroblasts from patients would provide a valuable tool in the study of the disease as it would present a readily accessible and controllable system for examination. Furthermore, fibroblast expression would allow diagnosis of fetal and presumptomatic cases. An unusual staining pattern of MMD cultured skin fibroblasts as seen by light microscopy, namely, an increase in alcianophilia and metachromasia, has been reported; both these techniques suggest an altered glycosaminoglycan metabolism An altered growth pattern has also been described. One reference on cultured skin fibroblasts from a different dystrophy (Duchenne Muscular Dystrophy) reports increased cytoplasmic inclusions seen by electron microscopy. Also, ultrastructural alterations have been reported in muscle and thalamus biopsies from MMD patients, but no electron microscopical data is available on MMD cultured skin fibroblasts.

Structure and Interaction of Protamine by Dark Field Electron Microscopy

1976 ◽  
Vol 34 ◽  
pp. 160-161
Author(s):  
D.P. Bazett-Jones ◽  
F.P. Ottensmeyer
Keyword(s):  
Electron Microscopy ◽  
Dark Field ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
3 Dimensional ◽  
Field Electron ◽  
Proposed Model ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Positively Charged

It has been shown for some time that it is possible to obtain images of small unstained proteins, with a resolution of approximately 5Å using dark field electron microscopy (1,2). Applying this technique, we have observed a uniformity in size and shape of the 2-dimensional images of pure specimens of fish protamines (salmon, herring (clupeine, Y-l) and rainbow trout (Salmo irideus)). On the basis of these images, a model for the 3-dimensional structure of the fish protamines has been proposed (2).The known amino acid sequences of fish protamines show stretches of positively charged arginines, separated by regions of neutral amino acids (3). The proposed model for protamine structure (2) consists of an irregular, right-handed helix with the segments of adjacent arginines forming the loops of the coil.

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