Cooperative regulation of myosin-S1 binding to actin filaments by a continuous flexible Tm–Tn chain

The polarity of actin filaments is fundamental for the subcellular mechanics of actin-myosin interaction; however, little is known about how actin filaments are oriented with respect to myosin in non-muscle cells and how actin polarity organization is established and maintained. Here we approach these questions by investigating changes in the organization and polarity of actin relative to myosin II during actin filament translocation. Actin and myosin II reorganization was followed both kinetically, using microinjected fluorescent analogs of actin and myosin, and ultrastructurally, using myosin S1 decoration and immunogold labelling, in cultured fibroblasts that were induced to contract by treatment with cytochalasin D. We observed rapid (within 15 minutes) formation of ordered actin filament arrays: short tapered bundles and aster-like assemblies, in which filaments had uniform polarity with their barbed ends oriented toward the aggregate of myosin II at the base of a bundle or in the center of an aster. The resulting asters further interacted with each other and aggregated into bigger asters. The arrangement of actin in asters was in sharp contrast to the mixed polarity of actin filaments relative to myosin in non-treated cells. At the edge of the cell, actin filaments became oriented with their barbed ends toward the cell center; that is, the orientation was opposite to what was observed at the edge of nontreated cells. This rearrangement is indicative of relative translocation of actin and myosin II and of the ability of myosin II to sort actin filaments with respect to their polarity during translocation. The results suggest that the myosin II-actin system of non-muscle cells is organized as a dynamic network where actin filament arrangement is defined in the course of its interaction with myosin II.

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The variable twist of actin and its modulation by actin-binding proteins

The Journal of Cell Biology ◽

10.1083/jcb.104.4.1005 ◽

1987 ◽

Vol 104 (4) ◽

pp. 1005-1017 ◽

Cited By ~ 55

Author(s):

DL Stokes ◽

DJ DeRosier

Keyword(s):

Actin Filaments ◽

Binding Proteins ◽

Biological Function ◽

Fourier Transforms ◽

Actin Binding ◽

Filament Length ◽

Layer Line ◽

Actin Binding Proteins ◽

Thin Filaments ◽

Myosin S1

Previous studies demonstrated that actin filaments have variable twist in which the intersubunit angles vary by approximately +/- 10 degrees within a filament. In this work we show that this variability was unchanged when different methods were used to prepare filaments for electron microscopy. We also show that actin-binding proteins can modulate the variability in twist. Three preparations of actin filaments were photographed in the electron microscope: negatively stained filaments, replicas of rapidly frozen, etched filaments, and frozen hydrated filaments. In addition, micrographs of actin + tropomyosin + troponin (thin filaments), of actin + myosin S1 (decorated filaments), and of filaments frayed from the acrosomal process of Limulus sperm (Limulus filaments) were obtained. We used two independent methods to measure variable twist based on Fourier transforms of single filaments. The first involved measuring layer line intensity versus filament length and the second involved measuring layer line position. We measured a variability in the intersubunit angle of actin filaments of approximately 12 degrees independent of the method of preparation or of measurement. Thin filaments have 15 degrees of variability, but the increase over pure actin is not statistically significant. Decorated filaments and Limulus filaments, however, have significantly less variability (approximately 2 and 1 degree, respectively), indicating a torsional stiffening relative to actin. The results from actin alone using different preparative methods are evidence that variable twist is a property of actin in solution. The results from actin filaments in the presence of actin-binding proteins suggest that the angular variability can be modulated, depending on the biological function.

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Force Spectroscopy Reveals Multiple “Closed States” of the Muscle Thin Filament

Journal of Biological Chemistry ◽

10.1074/jbc.m110.167957 ◽

2011 ◽

Vol 286 (27) ◽

pp. 24135-24141 ◽

Cited By ~ 8

Author(s):

Vijay S. Rao ◽

Amy M. Clobes ◽

William H. Guilford

Keyword(s):

Actin Filaments ◽

Thin Filament ◽

Striated Muscle ◽

Critical Role ◽

Laser Trap ◽

Myosin S1 ◽

State Affect ◽

Myosin Binding ◽

Three State Model ◽

Muscle Thin Filament

Tropomyosin (Tm) plays a critical role in regulating the contraction of striated muscle. The three-state model of activation posits that Tm exists in three positions on the thin filament: “blocked” in the absence of calcium when myosin cannot bind, “closed” when calcium binds troponin and Tm partially covers the myosin binding site, and “open” after myosin binding forces Tm completely off neighboring sites. However, we recently showed that actin filaments decorated with phosphorylated Tm are driven by myosin with greater force than bare actin filaments. This result cannot be explained by simple steric hindrance and suggests that Tm may have additional effects on actin-myosin interactions. We therefore tested the hypothesis that Tm and its phosphorylation state affect the rate at which single actin-myosin bonds form and rupture. Using a laser trap, we measured the time necessary for the first bond to form between actin and rigor heavy meromyosin and the load-dependent durations of those bonds. Measurements were repeated in the presence of subsaturating myosin-S1 to force Tm from the closed to the open state. Maximum bond lifetimes increased in the open state, but only when Tm was phosphorylated. While the frequency with which bonds formed was extremely low in the closed state, when a bond did form it took significantly less time to do so than with bare actin. These data suggest there are at least two closed states of the thin filament, and that Tm provides additional points of contact for myosin.

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Three-dimensional arrangement of F-actin in the contractile ring of fission yeast

The Journal of Cell Biology ◽

10.1083/jcb.200612018 ◽

2007 ◽

Vol 178 (5) ◽

pp. 765-771 ◽

Cited By ~ 142

Author(s):

Tomoko Kamasaki ◽

Masako Osumi ◽

Issei Mabuchi

Keyword(s):

Electron Microscopy ◽

Fission Yeast ◽

Actin Filaments ◽

Three Dimensional ◽

Single Site ◽

Yeast Cells ◽

Contractile Ring ◽

Division Plane ◽

Early Stages ◽

Myosin S1

The contractile ring, which is required for cytokinesis in animal and yeast cells, consists mainly of actin filaments. Here, we investigate the directionality of the filaments in fission yeast using myosin S1 decoration and electron microscopy. The contractile ring is composed of around 1,000 to 2,000 filaments each around 0.6 μm in length. During the early stages of cytokinesis, the ring consists of two semicircular populations of parallel filaments of opposite directionality. At later stages, before contraction, the ring filaments show mixed directionality. We consider that the ring is initially assembled from a single site in the division plane and that filaments subsequently rearrange before contraction initiates.

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Mechanism of brush border contractility studied by the quick-freeze, deep-etch method.

The Journal of Cell Biology ◽

10.1083/jcb.96.5.1325 ◽

1983 ◽

Vol 96 (5) ◽

pp. 1325-1336 ◽

Cited By ~ 95

Author(s):

N Hirokawa ◽

T C Keller ◽

R Chasan ◽

M S Mooseker

Keyword(s):

Brush Border ◽

Actin Filaments ◽

Ultrastructural Changes ◽

Immunofluorescent Localization ◽

Terminal Web ◽

Myosin S1 ◽

Brush Borders ◽

Mixed Polarity ◽

The Individual ◽

Replication Technique

We have analyzed terminal web contraction in sheets of glycerinated chicken small intestine epithelium and in isolated intestinal brush borders using a quick-freeze, deep-etch, rotary shadow replication technique. In the presence of Mg-ATP at 37 degrees C, the terminal web region of each cell in the glycerinated sheet and of each isolated brush border became severely constricted at the level of its zonula adherens (ZA). Consequently, the individual brush borders rounded up, splaying out their microvilli in fanlike patterns. The most prominent ultrastructural changes that occurred during terminal web contraction were a dramatic decrease in the diameter of the circumferential ring composed of a bundle of 8-9-nm filaments adjacent to the zonula adherens and a decrease in the number of cross-linkers between the microvillus rootlets. Microvilli were not retracted into the terminal web. We have used myosin S1 decoration to demonstrate that most of the circumferential bundle filaments are actin and that the actin filaments are arranged in the bundle with mixed polarity. Some filaments within the bundle did not decorate with myosin S1 and had tiny projections that appeared to be attached to adjacent actin filaments. Because of their morphology and immunofluorescent localization of myosin within this region of the terminal web, we propose that these undecorated filaments are myosin. From these results, we conclude that brush border contraction is caused primarily by an active sliding of actin and myosin filaments within the circumferential bundle of filaments associated with the ZA.

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Electron Microscopy of Cytoplasmic Contractile Proteins

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100070333 ◽

1978 ◽

Vol 36 (3) ◽

pp. 606-614 ◽

Cited By ~ 1

Author(s):

T.D. Pollard ◽

P. Maupin

Keyword(s):

Electron Microscopy ◽

Actin Filaments ◽

Three Dimensional ◽

Uranyl Acetate ◽

Contractile Proteins ◽

Dimensional Structure ◽

X Ray Diffraction ◽

Cytoplasmic Matrix ◽

Computer Image Processing ◽

Nonmuscle Cells

In this paper we review some of the contributions that electron microscopy has made to the analysis of actin and myosin from nonmuscle cells. We place particular emphasis upon the limitations of the ultrastructural techniques used to study these cytoplasmic contractile proteins, because it is not widely recognized how difficult it is to preserve these elements of the cytoplasmic matrix for electron microscopy. The structure of actin filaments is well preserved for electron microscope observation by negative staining with uranyl acetate (Figure 1). In fact, to a resolution of about 3nm the three-dimensional structure of actin filaments determined by computer image processing of electron micrographs of negatively stained specimens (Moore et al., 1970) is indistinguishable from the structure revealed by X-ray diffraction of living muscle.

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Destruction of Actin Filaments by Osmium Tetroxide

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079772 ◽

1977 ◽

Vol 35 ◽

pp. 466-467

Author(s):

P. Maupin-Szamier ◽

T. D. Pollard

Keyword(s):

Actin Filaments ◽

Time Course ◽

Osmium Tetroxide ◽

Rabbit Muscle ◽

Muscle Actin ◽

Sodium Phosphate Buffer ◽

Transmission Electron ◽

The Difference ◽

Rates Of Decay ◽

Short Time

We have studied the destruction of rabbit muscle actin filaments by osmium tetroxide (OSO4) to develop methods which will preserve the structure of actin filaments during preparation for transmission electron microscopy.Negatively stained F-actin, which appears as smooth, gently curved filaments in control samples (Fig. 1a), acquire an angular, distorted profile and break into progressively shorter pieces after exposure to OSO4 (Fig. 1b,c). We followed the time course of the reaction with viscometry since it is a simple, quantitative method to assess filament integrity. The difference in rates of decay in viscosity of polymerized actin solutions after the addition of four concentrations of OSO4 is illustrated in Fig. 2. Viscometry indicated that the rate of actin filament destruction is also dependent upon temperature, buffer type, buffer concentration, and pH, and requires the continued presence of OSO4. The conditions most favorable to filament preservation are fixation in a low concentration of OSO4 for a short time at 0°C in 100mM sodium phosphate buffer, pH 6.0.

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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.

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Orientation of actin filaments during motion in motility assay

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136635 ◽

1995 ◽

Vol 53 ◽

pp. 52-53

Author(s):

J. Borejdo ◽

S. Burlacu

Keyword(s):

Actin Filaments ◽

Thin Filament ◽

Striated Muscle ◽

Myosin Head ◽

Classical Method ◽

Polarization Of Fluorescence ◽

Single Protein ◽

Intracellular Organelles ◽

Change Orientation ◽

Active Entity

Polarization of fluorescence is a classical method to assess orientation or mobility of macromolecules. It has been a common practice to measure polarization of fluorescence through a microscope to characterize orientation or mobility of intracellular organelles, for example anisotropic bands in striated muscle. Recently, we have extended this technique to characterize single protein molecules. The scientific question concerned the current problem in muscle motility: whether myosin heads or actin filaments change orientation during contraction. The classical view is that the force-generating step in muscle is caused by change in orientation of myosin head (subfragment-1 or SI) relative to the axis of thin filament. The molecular impeller which causes this change resides at the interface between actin and SI, but it is not clear whether only the myosin head or both SI and actin change orientation during contraction. Most studies assume that observed orientational change in myosin head is a reflection of the fact that myosin is an active entity and actin serves merely as a passive "rail" on which myosin moves.

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Modulated polarization microscopy displays the dynamics of cytoskeletal structures in living, motile cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140889 ◽

1995 ◽

Vol 53 ◽

pp. 902-903

Author(s):

J. R. Kuhn ◽

M. Poenie

Keyword(s):

Mitotic Spindle ◽

Actin Filaments ◽

Cell Shape ◽

Dynamic Structure ◽

Living Cells ◽

Cytoskeletal Proteins ◽

Polarization Microscopy ◽

Video Enhancement ◽

Ultrastructural Studies ◽

Motile Cells

Cell shape and movement are controlled by elements of the cytoskeleton including actin filaments an microtubules. Unfortunately, it is difficult to visualize the cytoskeleton in living cells and hence follow it dynamics. Immunofluorescence and ultrastructural studies of fixed cells while providing clear images of the cytoskeleton, give only a static picture of this dynamic structure. Microinjection of fluorescently Is beled cytoskeletal proteins has proved useful as a way to follow some cytoskeletal events, but long terry studies are generally limited by the bleaching of fluorophores and presence of unassembled monomers.Polarization microscopy has the potential for visualizing the cytoskeleton. Although at present, it ha mainly been used for visualizing the mitotic spindle. Polarization microscopy is attractive in that it pro vides a way to selectively image structures such as cytoskeletal filaments that are birefringent. By combing ing standard polarization microscopy with video enhancement techniques it has been possible to image single filaments. In this case, however, filament intensity depends on the orientation of the polarizer and analyzer with respect to the specimen.

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