Modulators of actin-myosin dissociation: basis for muscle type functional differences during fatigue

The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define “fatigability” in vivo of fast vs. slow myosin isoforms, at the molecular level, are not yet fully understood. We examined the modulation of the ATP-induced myosin subfragment 1 (S1) dissociation from pyrene-actin by inorganic phosphate (Pi), pH, and temperature using a specially modified stopped-flow system that allowed fast kinetics measurements at physiological temperature. We contrasted the properties of rabbit psoas (fast) and bovine masseter (slow) myosins (obtained from samples collected from New Zealand rabbits and from a licensed abattoir, respectively, according to institutional and national ethics permits). To identify ATP cycling biochemical intermediates, we assessed ATP binding to a preequilibrated mixture of actomyosin and variable [ADP], pH (pH 7 vs. pH 6.2), and Pi (zero, 15, or 30 added mM Pi) in a range of temperatures (5 to 45°C). Temperature and pH variations had little, if any, effect on the ADP dissociation constant ( KADP) for fast S1, but for slow S1, KADP was weakened with increasing temperature or low pH. In the absence of ADP, the dissociation constant for phosphate ( KPi) was weakened with increasing temperature for fast S1. In the presence of ADP, myosin type differences were revealed at the apparent phosphate affinity, depending on pH and temperature. Overall, the newly revealed kinetic differences between myosin types could help explain the in vivo observed muscle type functional differences at rest and during fatigue.

Download Full-text

Myosin VI Stabilizes an Actin Network during Drosophila Spermatid Individualization

Molecular Biology of the Cell ◽

10.1091/mbc.e06-01-0031 ◽

2006 ◽

Vol 17 (6) ◽

pp. 2559-2571 ◽

Cited By ~ 62

Author(s):

Tatsuhiko Noguchi ◽

Marta Lenartowska ◽

Kathryn G. Miller

Keyword(s):

Green Fluorescent Protein ◽

Actin Filaments ◽

Fluorescent Protein ◽

Cross Linking ◽

Myosin Vi ◽

Actin Network ◽

Myosin Subfragment ◽

Myosin Subfragment 1 ◽

Green Fluorescent

Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells.

Download Full-text

Polymerization of actin. VI. The polarity of the actin filaments in the acrosomal process and how it might be determined.

The Journal of Cell Biology ◽

10.1083/jcb.81.3.608 ◽

1979 ◽

Vol 81 (3) ◽

pp. 608-623 ◽

Cited By ~ 43

Author(s):

L G Tilney ◽

N Kallenbach

Keyword(s):

Low Temperature ◽

Actin Filaments ◽

Critical Concentration ◽

Actin Monomer ◽

Spontaneous Nucleation ◽

Myosin Subfragment ◽

Acrosomal Reaction ◽

Myosin Subfragment 1

The polarity of the actin filaments which assemble from the nucleating body or actomere of Thyone and Pisaster sperm was determined using myosin subfragment 1 decoration. The polarity was found to be unidirectional with the arrowheads pointing towards the cell center. When polymerization is induced at low temperature with concentrations of actin near the critical concentration for polymerization, elongation of filaments occurs preferentially off the apical end. If the sperm are induced to undergo the acrosomal reaction with an ionophore, the polarity of the actin filaments attached to the actomere is the same as that already described, but the filaments which polymerize parallel to, but peripheral to, those extending from the actomere are randomly polarized. These randomly polarized filaments appear to result from spontaneous nucleation. When sperm are induced to undergo the acrosomal reaction with eggs, the polarity of the actin filaments is also unidirectional with the arrowheads pointing towards the cell center. From these results we conclude: (a) that the actomere, by nucleating the polymerization of actin filaments, controls the polarity of the actin filaments in the acrosomal process, (b) that the actomere recognizes a surface of the actin monomer that is different from that surface recognized by the dense material attached to membranes, and (c) that egg myosin could not act to pull the sperm into the egg. Included is a discussion of how the observation that monomers add largely to one end of a decorated filament in vitro relates to these in vivo observations.

Download Full-text

Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102869 ◽

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.

Download Full-text