scholarly journals Binding sites of calmodulin and actin on the brain spectrin, calspectin.

1983 ◽  
Vol 97 (2) ◽  
pp. 574-578 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
H Ishikawa ◽  
M Kurokawa ◽  
K Morimoto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Complex Formation ◽  
Binding Sites ◽  
Actin Binding ◽  
Calmodulin Binding ◽  
20 Nm ◽  
Labeling Method ◽  
The Brain ◽  
Brain Spectrin ◽  
Rotary Shadowing

We used rotary-shadowing electron microscopy to map the calmodulin-and actin-binding sites on the brain spectrin, calspectin (or fodrin). Calspectin dimers appeared as rods 110 nm long and joined in a head-to-head manner to form tetramers 220 nm long. We determined calmodulin-binding sites by a ferritin-labeling method combined with biotin-avidin complex formation. Ferritin particles were found to attach to the head parts of calspectin dimers at a position 10-20 nm from the top of the head. The number of the calmodulin-binding sites seemed to be only one for each dimer and two for each tetramer. In contrast, the actin-binding sites were localized at the tail ends of the calspectin molecules. The tetramers attached to muscle F-actin with their tail ends and often cross-linked adjacent filaments. The results are discussed in view of the analogy to the erythrocyte spectrin.

scholarly journals Ultracytochemistry of calmodulin binding sites in myocardial cells by staining of frozen thin sections with colloidal gold-labeled calmodulin.

1989 ◽  
Vol 37 (2) ◽  
pp. 249-256 ◽  
Author(s):  
K Fujimoto ◽  
N Araki ◽  
K S Ogawa ◽  
S Kondo ◽  
T Kitaoka ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Biologically Active ◽  
Myocardial Cells ◽  
Thin Sections ◽  
Gold Particles ◽  
Calmodulin Binding ◽  
Tissue Sections

Calmodulin (CaM) has been implicated as a multifunctional regulator of Ca2+ in the cytoplasm of cells. We have recently introduced biologically active colloidal gold-labeled CaM as a marker for identifying potential CaM binding sites (unoccupied by endogenous CaM at the time of fixation) by electron microscopy and have stained frozen thin sections of rat cardiac muscle with this conjugate. In the presence of Ca2+, gold particles indicating CaM binding sites were found localized on the sarcoplasmic reticulum, mitochondria, and gap junctions. Control tissue sections treated with EGTA or exposed to excess amounts of unlabeled native CaM before staining showed no binding. We believe that cytochemistry of potential CaM binding sites revealed by staining with labeled exogenous CaM is useful in correlating known biochemical reactions of CaM with particular cell activities.

scholarly journals Structural Evidence that the P/Q Domain of ZipA Is an Unstructured, Flexible Tether between the Membrane and the C-Terminal FtsZ-Binding Domain

2002 ◽  
Vol 184 (15) ◽  
pp. 4313-4315 ◽  
Author(s):  
Tomoo Ohashi ◽  
Cynthia A. Hale ◽  
Piet A. J. de Boer ◽  
Harold P. Erickson
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
Transmembrane Domain ◽  
Persistence Length ◽  
Binding Domain ◽  
Globular Domain ◽  
Cell Division Protein ◽  
20 Nm ◽  
Rotary Shadowing

ABSTRACT The cell division protein ZipA has an N-terminal transmembrane domain and a C-terminal globular domain that binds FtsZ. Between them are a charged domain and a P/Q domain rich in proline and glutamine that has been proposed to be an unfolded polypeptide. Here we provide evidence obtained by electron microscopy that the P/Q domain is a flexible tether ranging in length from 8 to 20 nm and invisible in rotary shadowing electron microscopy. We estimated a persistence length of 0.66 nm, which is similar to the persistence lengths of other unfolded and unstructured polypeptides.

scholarly journals Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy

1994 ◽  
Vol 67 (6) ◽  
pp. 2286-2295 ◽  
Author(s):  
T. Wagenknecht ◽  
J. Berkowitz ◽  
R. Grassucci ◽  
A.P. Timerman ◽  
S. Fleischer
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Ryanodine Receptor ◽  
Binding Sites ◽  
Calmodulin Binding

scholarly journals Structural comparison of several actin-binding macromolecules.

1980 ◽  
Vol 85 (2) ◽  
pp. 489-495 ◽  
Author(s):  
J M Tyler ◽  
J M Anderson ◽  
D Branton
Keyword(s):  
Electron Microscopy ◽  
Binding Protein ◽  
Actin Binding ◽  
Erythrocyte Membranes ◽  
Structural Comparison ◽  
Flexible Molecules ◽  
Actin Binding Protein ◽  
Rotary Shadowing

The cytoskeletal components, macrophage actin-binding protein and filamin, were dried from glycerol and examined by low-angle rotary shadowing electron microscopy. Both are elongate, flexible molecules whose general morphologi is similar to that of erythrocyte spectrin. Neither actin-binding protein nor filamin binds to spectrin-depleted erythrocyte membranes.

scholarly journals Accumulation and Effect of Silver Nanoparticles Functionalized with Spirulina platensis on Rats

Nanomaterials ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 2992
Author(s):  
Ludmila Rudi ◽  
Inga Zinicovscaia ◽  
Liliana Cepoi ◽  
Tatiana Chiriac ◽  
Alexandra Peshkova ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Silver Nanoparticles ◽  
Spirulina Platensis ◽  
Biochemical Tests ◽  
Hematological Indices ◽  
Transmission Electron ◽  
The Difference ◽  
20 Nm ◽  
And Control ◽  
The Brain

The effect of unmodified and functionalized Spirulina platensis biomass silver nanoparticles on rats during prolonged oral administration was assessed. Silver nanoparticles were characterized by using transmission electron microscopy, while their uptake by the biomass was confirmed using scanning electron microscopy and energy dispersive analysis. The content of silver in the different organs of rats after a period of administration (28 days) or after an additional clearance period (28 days) was ascertained by using neutron activation analysis. In animals administrated with the unmodified nanoparticles, the highest content of silver was determined in the brain and kidneys, while in animals administrated with AgNP-Spirulina, silver was mainly accumulated in the brain and testicles. After the clearance period, silver was excreted rapidly from the spleen and kidneys; however, the excretion from the brain was very low, regardless of the type of nanoparticles. Hematological and biochemical tests were performed in order to reveal the effect of nanoparticles on rats. The difference in the content of eosinophils in the experimental and control groups was statistically significant. The hematological indices of the rats did not change significantly under the action of the silver nanoparticles except for the content of reticulocytes and eosinophils, which increased significantly. Changes in the biochemical parameters did not exceed the limits of normal values. Silver nanoparticles with the sizes of 8–20 nm can penetrate the blood–brain barrier, and their persistence after a period of clearance indicated the irreversibility of this process.

scholarly journals Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

2020 ◽  
Vol 6 (23) ◽  
pp. eaaz7651 ◽  
Author(s):  
Austin Zimmet ◽  
Trevor Van Eeuwen ◽  
Malgorzata Boczkowska ◽  
Grzegorz Rebowski ◽  
Kenji Murakami ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Motility ◽  
Binding Sites ◽  
Activation Mechanism ◽  
Actin Binding ◽  
Cross Linking ◽  
Specific Interactions ◽  
Related Protein ◽  
Actin Networks ◽  
Cryo Electron Microscopy

Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo–electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex.

scholarly journals Morphology of Isolated Gli349, a Leg Protein Responsible for Mycoplasma mobile Gliding via Glass Binding, Revealed by Rotary Shadowing Electron Microscopy

2006 ◽  
Vol 188 (8) ◽  
pp. 2821-2828 ◽  
Author(s):  
Jun Adan-Kubo ◽  
Atsuko Uenoyama ◽  
Toshiaki Arata ◽  
Makoto Miyata
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Gel Filtration ◽  
Solid Surfaces ◽  
Membrane Protrusion ◽  
Molecular Image ◽  
Peptide Length ◽  
20 Nm ◽  
Molecular Images ◽  
Rotary Shadowing

ABSTRACT Several species of mycoplasmas rely on an unknown mechanism to glide across solid surfaces in the direction of a membrane protrusion at the cell pole. Our recent studies on the fastest species, Mycoplasma mobile, suggested that a 349-kDa protein, Gli349, localized at the base of the membrane protrusion called the neck, forms legs that stick out from the neck and propel the cell by repeatedly binding to and releasing from a solid surface, based on the energy of ATP hydrolysis. Here, the Gli349 protein was isolated from mycoplasma cells and its structure was analyzed. Gel filtration analysis showed that the isolated Gli349 protein is monomeric. Rotary shadowing electron microscopy revealed that the molecular structure resembles the symbol for an eighth note in music. It contains an oval foot 14 nm long in axis. From this foot extend three rods in tandem of 43, 20, and 20 nm, in that order. The hinge connecting the first and second rods is flexible, while the next hinge has a distinct preference in its angle, near 90 degrees. Molecular images revealed that a monoclonal antibody that can bind to the position at one-third of the total peptide length from the N terminus bound to a position two-thirds from the foot end, suggesting that the foot corresponds to the C-terminal region. The amino acid sequence was assigned to the molecular image, and the topology of the molecule in the gliding machinery is discussed.

Application of Scanning Electron Microscopy to Medical Research

1969 ◽  
Vol 27 ◽  
pp. 12-13
Author(s):  
J. D. Hutchison
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Medical Research ◽  
Prosthetic Heart Valve ◽  
School Of Medicine ◽  
Transmission Electron ◽  
Definition Of ◽  
Mechanism Of Failure ◽  
The Brain ◽  
Scanning Electron

When the transmission electron microscope was commercially introduced a few years ago, it was heralded as one of the most significant aids to medical research of the century. It continues to occupy that niche; however, the scanning electron microscope is gaining rapidly in relative importance as it fills the gap between conventional optical microscopy and transmission electron microscopy.IBM Boulder is conducting three major programs in cooperation with the Colorado School of Medicine. These are the study of the mechanism of failure of the prosthetic heart valve, the study of the ultrastructure of lung tissue, and the definition of the function of the cilia of the ventricular ependyma of the brain.

Sites of Insulin Action Using Ferritin Labeling

1971 ◽  
Vol 29 ◽  
pp. 540-541
Author(s):  
Burton B. Silver ◽  
Ronald S. Nelson
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Anterior Pituitary ◽  
Insulin Action ◽  
Diabetic Rats ◽  
Insulin Antibody ◽  
Fat Pad ◽  
Target Organs ◽  
Uranium Salts ◽  
Sugar Levels

Some investigators feel that insulin does not enter cells but exerts its influence in some manner on the cell surface. Ferritin labeling of insulin and insulin antibody was used to determine if binding sites of insulin to specific target organs could be seen with electron microscopy.Alloxanized rats were considered diabetic if blood sugar levels were in excess of 300 mg %. Test reagents included ferritin, ferritin labeled insulin, and ferritin labeled insulin antibody. Target organs examined were were diaphragm, kidney, gastrocnemius, fat pad, liver and anterior pituitary. Reagents were administered through the left common carotid. Survival time was at least one hour in test animals. Tissue incubation studies were also done in normal as well as diabetic rats. Specimens were fixed in gluteraldehyde and osmium followed by staining with lead and uranium salts. Some tissues were not stained.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

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