Studies on the motility of the foraminifera. I. Ultrastructure of the reticulopodial network of Allogromia laticollaris (Arnold).

Allogromia laticollaris, a benthic marine foraminifer, extends numerous trunk filopodia that repeatedly branch, anastomose, and fuse again to form the reticulopodial network (RPN), within which an incessant streaming of cytoplasmic particles occurs. The motion of the particles is saltatory and bidirectional, even in the thinnest filopodia detected by optical microscopy. Fibrils are visible by differential interference microscopy, and the PRN displays positive birefringence in polarized light. These fibrils remain intact after lysis and extraction of the RPN in solutions that stabilize microtubules (MTs). Electron micrographs of thin sections through these lysed and stabilized cytoskeletal models reveal bundles of MTs. The RPNs of living Allogromia may be preserved by standard EM fixatives only after acclimatization to calcium-free seawater, in which the streaming is normal. The MTs in the RPN are typically arranged in bundles that generally lie parallel to the long axis of the trunk and branch filopodia. Stereo electron micrographs of whole-mount, fixed, and critical-point-dried organisms show that the complex pattern of MT deployment reflects the pattern of particle motion in both flattened and highly branched portions of the RPN. Cytoplasmic particles, some of which have a fuzzy coat, are closely associated with, and preferentially oriented along, either single MTs or MT bundles. Thin filaments (approximately 5 nm) are also observed within the network, lying parallel to and interdigitating with the MTs, and in flattened terminal areas of the filopodia. These filaments do not bind skeletal muscle myosin S1 under conditions that heavily decorate actin filaments in controls (human blood platelets), and are approximately 20% too thin to be identified ultrastructurally as F-actin.

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Non-uniform staining of myofibrila bands by a monoclonal antibody to skeletal muscle myosin S1 heavy chain

Tissue and Cell ◽

10.1016/0040-8166(83)90067-8 ◽

1983 ◽

Vol 15 (3) ◽

pp. 341-349 ◽

Cited By ~ 5

Author(s):

P. Wachsberger ◽

L. Lampson ◽

F.A. Pepe

Keyword(s):

Skeletal Muscle ◽

Monoclonal Antibody ◽

Heavy Chain ◽

Skeletal Muscle Myosin ◽

Myosin S1 ◽

Muscle Myosin

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The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II

Journal of Biological Chemistry ◽

10.1074/jbc.m115.688002 ◽

2015 ◽

Vol 291 (4) ◽

pp. 1763-1773 ◽

Cited By ~ 10

Author(s):

Marieke J. Bloemink ◽

Girish C. Melkani ◽

Sanford I. Bernstein ◽

Michael A. Geeves

Keyword(s):

Kinetic Analysis ◽

Flight Muscle ◽

Fine Tuning ◽

Wild Type ◽

Skeletal Muscle Myosin ◽

Myosin S1 ◽

Recovery Stroke ◽

Atp Binding And Hydrolysis ◽

Muscle Myosin ◽

Hydrolysis Of

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.

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Troponin in embryonic chick skeletal muscle cell in vitro: an immunoelectron microscopic study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008167x ◽

1978 ◽

Vol 36 (2) ◽

pp. 162-163

Author(s):

Yutaka Shimada ◽

Takashi Obinata

Keyword(s):

Skeletal Muscle ◽

Chicken Breast ◽

Thin Filaments ◽

Structural Localization ◽

Adult Chicken ◽

Skeletal Muscle Myosin ◽

Ammonium Sulphate Fractionation ◽

The Right ◽

Muscle Myosin

In developing skeletal muscle, myosin and actin are synthesized and polymerized into filamentous forms, thick and thin filaments, respectively. These myofilaments of two varieties have been shown, with the use of "decoration with heavy meromyosin" technique, to be arranged at the right polarity and spatial position as those seen in mature myofibrils from the initial phases of myofibrillogenesis. The question arises as to whether regulatory proteins are distributed along embryonic thin filaments from such early stages of development. In order to clarify this problem, the fine structural localization of troponin was investigated by the use of immunoelectron microscopy.Troponin and its components (troponin C [TN-C], I [TN-I] and T [TN-T]) were prepared from adult chicken breast muscles. Rabbit antisera against each of these troponin components were prepared. The serum was fractioned by ethanol or ammonium sulphate fractionation. Myogenic cells from 12-day chick embryonic thigh muscles were grown in monolayer and, after 2-8 days in vitro, the cultures were treated as follows: (i) They were immersed in 50% glycerol. The suspension of separated thin filaments was obtained by gentle homogenizing the cells.

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Phosphorylation of the calcium ion-regulated thin filaments from vascular smooth muscle A new regulatory mechanism?

Biochemical Journal ◽

10.1042/bj1970127 ◽

1981 ◽

Vol 197 (1) ◽

pp. 127-139 ◽

Cited By ~ 28

Author(s):

M Walters ◽

S B Marston

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Thin Filament ◽

Sodium Dodecyl ◽

Regulatory System ◽

Specific Protein ◽

Maximum Activity ◽

Thin Filaments ◽

Skeletal Muscle Myosin ◽

Muscle Myosin

The thin filaments of vascular smooth muscle (pig aorta) contain a Ca2+-sensitive regulatory system that resembles troponin-tropomyosin [Marston, Trevett & Walters (1980) Biochem. J. 185, 355-365]. Our thin-filament preparations also contain enzymes that phosphorylate and dephosphorylate a specific protein. Initial rate of phosphorylation was 0.42 +/- 0.10 (95% confidence limits) mumol of Pi/min per g of thin filaments; half-maximal incorporation was obtained in 4 1/2 min, and a maximum of 1.8 +/- 0.1 mumol of Pi/g of thin filaments was incorporated after 40 min (conditions: 1 mM-MgATP, 60 mM-MgATP, 60 mM-KCl, 10 mM-imidazole, pH 7.0, 5 mM-MgCl2, 10 mM-NaN3, 0.5 mM-dithiothreitol, 0.1 mM-CaCl2, 25 degrees C). On gel electrophoresis in polyacrylamide (4-30% gradient)/0.25% sodium dodecyl sulphate gel over 75% of protein-bound phosphate was in a single protein of mol.wt. 21000. On electrophoresis in polyacrylamide (8%)/6 M-urea (pH 8.6) gel the phosphoprotein remained at the origin. Phosphorylation was associated with an increase in the concentration of high-affinity (K congruent to 10(6) M-1) Ca2+-binding sites from 0.8-1.5 to 6.3 mumol of Ca2+/g of thin filaments. Phosphorylation also changed the regulatory properties of the skeletal-muscle myosin-aorta thin-filament MgATPase; maximum activity was unaltered, but the phosphorylated thin filaments required only 0.36 microM-Ca2+ for half-activation compared with 2.7 microM-Ca2+ for unphosphorylated thin filaments. The possible regulatory role of thin-filament phosphorylation is discussed.

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The restoration of blurred electron micrographs

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142529 ◽

1986 ◽

Vol 44 ◽

pp. 180-181

Author(s):

Bridget Carragher ◽

David A. Bluemke ◽

Michael J. Potel ◽

Robert Josephs

Keyword(s):

Cross Section ◽

Electron Micrographs ◽

Relative Motion ◽

Finite Thickness ◽

Image Plane ◽

Helical Axis ◽

Thin Sections ◽

Structural Details

We have investigated the feasibility of restoring blurred electron micrographs. Two related problems have been considered; the restoration of images blurred as a result of relative motion between the specimen and the image plane, and the restoration of images which are rotationally blurred about an axis. Micrographs taken while the specimen is drifting result in images which are blurred in the direction of motion. An example of rotational blurring arises in micrographs of thin sections of helical particles viewed in cross section. The twist of the particle within the finite thickness of the section causes the image to appear rotationally blurred about the helical axis. As a result, structural details, particularly at large distances from the helical axis, will be obscured.

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On the Mechanism by which Cell Contact Induces the Release Reaction of Blood Platelets; the Effect of Cationic Polymers

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649347 ◽

1972 ◽

Vol 27 (01) ◽

pp. 121-133 ◽

Cited By ~ 14

Author(s):

P Massini ◽

E. F Lüscher

Keyword(s):

Human Blood ◽

Calcium Ions ◽

Blood Platelets ◽

Cell Contact ◽

Second Phase ◽

Cationic Polymers ◽

Release Reaction ◽

Per Se ◽

Human Blood Platelets

SummaryHuman blood platelets are aggregated by the basic polymers polylysine and DEAE- dextran. Under certain conditions a second phase of aggregation, concomitant with the release reaction, is elicited. The presence of ADP, calcium ions and a plasmatic cofactor within the primary aggregates are necessary for the induction of the release reaction. These experiments demonstrate that cell contact per se does not lead to a release reaction ; in order to become effective it must take place in the presence of ADP.

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Chromatographic Isolation of the LDH-Isoenzymes of Human Blood Platelets and an Investigation of Their Enzyme Kinetics

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651273 ◽

1968 ◽

Vol 20 (03/04) ◽

pp. 301-313 ◽

Cited By ~ 2

Author(s):

W Schneider ◽

K Schumacher ◽

B Thiede ◽

R Gross

Keyword(s):

Human Blood ◽

Blood Platelets ◽

Deae Cellulose ◽

Order Reaction ◽

Kinetic Properties ◽

Ldh Isoenzymes ◽

Kinetic Investigations ◽

Substrate Concentrations ◽

Chromatographic Isolation ◽

Human Blood Platelets

SummaryThe LDH-isoenzymes of human blood platelets show a distinct predominance of the isoenzymes 2 and 3 upon chromatography on DEAE-cellulose. Small amounts of LDH-1 are also present, while only traces of LDH-4 and -5 can be detected.Enzyme kinetic investigations of the principal isoenzymes LDH-1, -2 and -3 clearly show that the differences in inhibition constants with pyruvate as substrate which are demonstrable at 25° largely disappear at 37°. On the other hand, the differences among the isoenzymes in their affinity for pyruvate and lactate as substrate as well as in with respect to the optimal substrate concentrations of pyruvate are more marked at 37° than at 25°. Also, the type of inhibition found with lactate as substrate is increasingly the expression of a higher order reaction in going from LDH-1 to LDH-3. A dependence of the LDH distribution pattern upon the metabolism of the cell is discussed. A comparison of our results with thrombocytes with those of other workers with erythrocytes and leucocytes makes it unlikely that the LDH pattern is directly dependent upon the existence of an oxidative metabolism. Rather, the redox potential of the cell could be of importance for the nature of the pattern of isoenzymes and for their differing kinetic properties.

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Interaction of Vasopressin with Human Blood Platelets: Dependency on Mg2+

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661662 ◽

1986 ◽

Vol 56 (03) ◽

pp. 260-262 ◽

Cited By ~ 6

Author(s):

Isabella Roos ◽

Fabrizia Ferracin ◽

Alfred Pletscher

Keyword(s):

Phosphatidic Acid ◽

Human Blood ◽

Arginine Vasopressin ◽

Specific Binding ◽

Blood Platelets ◽

Bivalent Cations ◽

Platelet Membranes ◽

Maximal Rise ◽

Human Blood Platelets ◽

Stimulation Of

SummaryArginine-vasopressin (AVP) in the presence of Mg2+ but not in the absence of bivalent cations led to accumulation of [32P]-phosphatidic acid ([32P]-PA) in human blood platelets. Mg2+ also enhanced the specific binding of [3H]-AVP to intact platelets. The concentrations of the cation which enabled AVP to cause half maximal rise of [32P]-PA and those inducing half maximal [3H]-AVP-binding were of the same order. It is concluded that the stimulation of phosphatidyl inositide breakdown by AVP in presence of Mg2+ is at least partially due to a Mg2+-induced enhancement of specific AVP-binding to the platelet membranes.

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