scholarly journals Time-resolved electron microscopic analysis of the behavior of myosin heads on actin filaments after photolysis of caged ATP.

1993 ◽  
Vol 121 (5) ◽  
pp. 1053-1064 ◽  
Author(s):  
T Funatsu ◽  
E Kono ◽  
S Tsukita
Keyword(s):  
Actin Filaments ◽  
Double Helix ◽  
Microscopic Analysis ◽  
Electron Microscopic ◽  
Weakly Bound ◽  
Time Resolved ◽  
Caged Atp ◽  
Dissociation Equilibrium ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P3-1-(2-nitrophenyl)ethyl ester of ATP (caged ATP) was analyzed with a newly developed freezing system using liquid helium. Actin and S1 (100 microM each) formed a ropelike double-helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. At 15 ms after photolysis, the ropelike double helix was partially disintegrated. The number of S1 attached to actin filaments gradually decreased up to 35 ms after photolysis, and no more changes were detected from 35 to 200 ms. After depletion of ATP, the ropelike double helix was reformed. Taking recent analyses of actomyosin kinetics into consideration, we concluded that most S1 observed on actin filaments at 35-200 ms are so called "weakly bound S1" (S1.ATP or S1.ADP.Pi) and that the weakly bound S1 under a rapid association-dissociation equilibrium with actin filaments can be captured by electron microscopy by means of our newly developed freezing system. This enabled us to directly compare the conformation of weakly and strongly bound S1. Within the resolution of deep-etch replica technique, there were no significant conformational differences between weakly and strongly bound S1, and neither types of S1 showed any positive cooperativity in their binding to actin filaments. Close comparison revealed that the weakly and strongly bound S1 have different angles of attachment to actin filaments. As compared to strongly bound S1, weakly bound S1 showed a significantly broader distribution of attachment angles. These results are discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.

Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

1988 ◽  
Vol 46 ◽  
pp. 156-157
Author(s):  
Donald A. Winkelmann
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Actin Filaments ◽  
Light Chains ◽  
Myosin Filament ◽  
Primary Role ◽  
Heavy Chains ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Globular Head

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

Myosin subfragment 1 binding to relaxed actin filaments and steric model of relaxation

Biochemistry ◽  
1981 ◽  
Vol 20 (3) ◽  
pp. 641-649 ◽  
Author(s):  
John M. Murray ◽  
Annemarie Weber ◽  
Mary K. Knox
Keyword(s):  
Actin Filaments ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

scholarly journals Regulation of the acto·myosin subfragment 1 interaction by troponin/tropomyosin. Evidence for control of a specific isomerization between two acto·myosin subfragment 1 states

1991 ◽  
Vol 279 (3) ◽  
pp. 711-718 ◽  
Author(s):  
D F A McKillop ◽  
M A Geeves
Keyword(s):  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Thin Filaments ◽  
Closed State ◽  
Myosin Subfragment ◽  
Actomyosin Interaction ◽  
Binding Isotherms ◽  
Myosin Subfragment 1 ◽  
Site Binding

The co-operative binding of myosin subfragment 1 (S1) to reconstituted skeletal-muscle thin filaments has been examined by monitoring the fluorescence of a pyrene probe on Cys-374 of actin. The degree of co-operativity differs when phosphate, sulphate or ADP are bound to the S1 active site. Binding isotherms have been analysed according to the Geeves & Halsall [(1987) Biophys. J. 52, 215-220] model, which proposed that troponin and tropomyosin effected regulation of the actomyosin interaction by controlling an isomerization of the actomyosin complex. The data support the proposal that seven actin monomers associated with a single tropomyosin molecule act as a co-operative unit that can be in one of two states. In the ‘closed’ state myosin can bind to actin, but the subsequent isomerization is prevented. The isomerization is only allowed after the seven-actin unit is in the ‘open’ form. Ca2+ controls the proportion of actin filaments in the ‘closed’ and ‘open’ forms in the absence of myosin heads. The ratio of ‘closed’ to ‘open’ forms is approx. 50:1 in the absence of Ca2+ and 5:1 in its presence.

The dynamics of the interaction between myosin subfragment 1 and pyrene-labelled thin filaments, from rabbit skeletal muscle

1986 ◽  
Vol 229 (1254) ◽  
pp. 85-95 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Binding Affinity ◽  
Kinetic Studies ◽  
Rabbit Skeletal Muscle ◽  
Fluorescent Label ◽  
Weakly Bound ◽  
Thin Filaments ◽  
Fluorescence Titration ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

We have used actin labelled in Cys–374 with N -(1-pyrenyl)iodoacetamide to monitor the dynamics and equilibria of the interaction between myosin subfragment 1 and the actin–troponin–tropomyosin complex in the presence of calcium. These results are compared with those obtained for pure actin and myosin subfragment 1. The sensitivity of this fluorescent label allowed us to measure the binding affinity of myosin subfragment 1 for actin directly by fluorescence titration. The affinity of subfragment 1 for actin is increased sixfold by troponin–tropomyosin in the presence of calcium. Kinetic studies of the interaction of subfragment 1 and actin have revealed an isomerization of the actin–subfragment 1 complex from a state in which actin is weakly bound ( K a = 5.9 x 10 4 M -1 ) to a more tightly bound complex ( K a = 1.7 x 10 7 M -1 ) (Coates, Criddle & Geeves (1985) Biochem. J. 232, 351). Results in the presence of troponin–tropomyosin show the same isomerization. The sixfold increase in affinity of subfragment 1 for actin is shown to be due to a decrease in the rate of dissociation of actin from the weakly bound complex.

Inhibition of ATP Binding to Myofibrils and Acto-Myosin Subfragment 1 by Caged ATP

Biochemistry ◽  
1994 ◽  
Vol 33 (20) ◽  
pp. 6038-6042 ◽  
Author(s):  
John Sleep ◽  
Christian Herrmann ◽  
Tom Barman ◽  
Franck Travers
Keyword(s):  
Atp Binding ◽  
Caged Atp ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

scholarly journals The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane-associated filaments.

1978 ◽  
Vol 79 (3) ◽  
pp. 846-852 ◽  
Author(s):  
D A Begg ◽  
R Rodewald ◽  
L I Rebhun
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Intestinal Epithelial ◽  
Improved Method ◽  
Thin Sections ◽  
Myosin Subfragment ◽  
Brush Borders ◽  
Nonmuscle Cells ◽  
Myosin Subfragment 1 ◽  
Uniform Polarity

We have developed an improved method for visualizing actin filament polarity in thin sections. Myosin subfragment-1 (S-1)-decorated actin filaments display a dramatically enhanced arrowhead configuration when fixed in a medium which contains 0.2 % tannic acid. With the exception of brush borders from intestinal epithelial cells, the arrowhead periodicity of decorated filaments in a variety of nonmuscle cells is similar to that in isolated myofibrils. The periodicity of decorated filaments in brush borders is significantly smaller. Actin filaments which attach to membranes display a clear, uniform polarity, with the S-1 arrowheads pointing away from the plasma membrane, while those which comprise the stress fibers of myoblasts and CHO cells have antiparallel polarities. These observations are consistent with a sliding filament mechanism of cell motility.

Myosin subfragment-1 is sufficient to move actin filaments in vitro

Nature ◽  
1987 ◽  
Vol 328 (6130) ◽  
pp. 536-539 ◽  
Author(s):  
Yoko Yano Toyoshima ◽  
Stephen J. Kron ◽  
Elizabeth M. McNally ◽  
Kenneth R. Niebling ◽  
Chikashi Toyoshima ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

scholarly journals A membrane cytoskeleton from Dictyostelium discoideum. III. Plasma membrane fragments bind predominantly to the sides of actin filaments.

1984 ◽  
Vol 99 (1) ◽  
pp. 71-78 ◽  
Author(s):  
C M Goodloe-Holland ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Dictyostelium Discoideum ◽  
Electron Micrographs ◽  
Actin Filaments ◽  
Lateral Movement ◽  
Heavy Meromyosin ◽  
Membrane Cytoskeleton ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

The binding between sonicated Dictyostelium discoideum plasma membrane fragments and F-actin on Sephacryl S-1000 beads was found to be competitively inhibited by myosin subfragment-1. This inhibition is MgATP-sensitive, exhibits a Ki of approximately 5 X 10(-8) M, and is reciprocal, since membranes inhibit the binding of 125I-heavy meromyosin to F-actin on beads. These experiments demonstrate that membrane binding and S-1 binding to F-actin on beads are mutually exclusive and, therefore, that the membrane fragments bind predominantly to the sides, rather than to the ends, of the actin filaments. This conclusion is supported by electron micrographs that show many lateral associations between membrane fragments and bead-associated actin filaments. Such lateral associations could play an important role in the organization and lateral movement of membrane proteins by the cytomusculature.

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