Structure of kinetochore fibres in crane-fly spermatocytes after irradiation with an ultraviolet microbeam: Neither microtubules nor actin filaments remain in the irradiated region

2003 ◽  
Vol 56 (3) ◽  
pp. 173-192 ◽  
Author(s):  
Arthur Forer ◽  
Tim Spurck ◽  
Jeremy D. Pickett-Heaps ◽  
Paula J. Wilson
Keyword(s):  
Actin Filaments ◽  
Ultraviolet Microbeam

scholarly journals ANALYSIS OF MUSCLE CONTRACTION BY ULTRAVIOLET MICROBEAM DISRUPTION OF SARCOMERE STRUCTURE

1965 ◽  
Vol 25 (2) ◽  
pp. 129-139 ◽  
Author(s):  
R. E. Stephens
Keyword(s):  
Muscle Contraction ◽  
Actin Filaments ◽  
Dry Mass ◽  
Folding Model ◽  
Microbeam Irradiation ◽  
Ultraviolet Microbeam ◽  
Apparent Loss ◽  
Myosin Filaments ◽  
Sliding Filament Theory

In an effort to differentiate between the sliding filament theory for muscle contraction and alternative views which propose attachment between actin and myosin filaments at or across the H zone, rabbit psoas myofibrils were irradiated in various areas of the sarcomere with an ultraviolet microbeam. Irradiation of the I band appears to destroy the actin filaments; in vitro irradiation of F actin causes an irreversible depolymerization of the protein. Irradiation of the A band disorients the myosin but causes no apparent loss of dry mass. These effects are maximal at the wavelength of maximum absorption of the proteins involved. Actin filaments, released at the Z line of a sarcomere, are seen to slide into the A band on addition of ATP. Irradiation of a full A band prevents contraction, whereas irradiation of two-thirds of the A band, leaving a lateral edge intact, permits contraction at the non-irradiated edge. Thus contraction can occur in what is in essence only one-third of a sarcomere, eliminating any necessity for postulated H zone connections. These observations are in complete accord with the classical sliding filament theory but incompatible with either the contralateral filament hypothesis or the actin folding model for muscle contraction.

Electron Microscopy of Cytoplasmic Contractile Proteins

1978 ◽  
Vol 36 (3) ◽  
pp. 606-614 ◽  
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.

Destruction of Actin Filaments by Osmium Tetroxide

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.

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.

Orientation of actin filaments during motion in motility assay

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.

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

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.

3-D examination of the cytoskeletal sheets of mammalian eggs

1992 ◽  
Vol 50 (1) ◽  
pp. 914-915
Author(s):  
Carolyn A. Larabell ◽  
David G. Capco ◽  
G. Ian Gallicano ◽  
Robert W. McGaughey ◽  
Karsten Dierksen ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Somatic Cells ◽  
Freeze Fracture ◽  
Blastocyst Stage ◽  
Cell Cytoplasm ◽  
Planar Structures ◽  
Cytoskeletal Network ◽  
Entire Cell ◽  
Cytoskeletal Networks ◽  
Mammalian Eggs

Mammalian eggs and embryos contain an elaborate cytoskeletal network of “sheets” which are distributed throughout the entire cell cytoplasm. Cytoskeletal sheets are long, planar structures unlike the cytoskeletal networks typical of somatic cells (actin filaments, microtubules, and intermediate filaments), which are filamentous. These sheets are not found in mammalian somatic cells nor are they found in nonmammalian eggs or embryos. Evidence that they are, indeed, cytoskeletal in nature is derived from studies demonstrating that 1) the sheets are retained in the detergent-resistant cytoskeleton fraction; 2) there are no associated membranes (determined by freeze-fracture); and 3) the sheets dissociate into filaments at the blastocyst stage of embryogenesis. Embedment-free sections of hamster eggs viewed at 60 kV show sheets running across the egg cytoplasm (Fig. 1). Although this approach provides excellent global views of the sheets and their reorganization during development, the mechanism of image formation for embedment-free sections does not permit evaluation of the sheets at high resolution.

Ultrastructural studies of the relationship between actin filaments and secretory granules

1990 ◽  
Vol 48 (3) ◽  
pp. 458-459
Author(s):  
Ellen Holm Nielsen
Keyword(s):  
Electron Microscopy ◽  
Colloidal Gold ◽  
Actin Filaments ◽  
Secretory Granules ◽  
Secretory Cells ◽  
Ultrastructural Studies ◽  
Transmission Electron ◽  
Granule Membrane ◽  
Ultrathin Sections ◽  
Lowicryl K4m

In secretory cells a dense and complex network of actin filaments is seen in the subplasmalemmal space attached to the cell membrane. During exocytosis this network is undergoing a rearrangement facilitating access of granules to plasma membrane in order that fusion of the membranes can take place. A filamentous network related to secretory granules has been reported, but its structural organization and composition have not been examined, although this network may be important for exocytosis.Samples of peritoneal mast cells were frozen at -70°C and thawed at 4°C in order to rupture the cells in such a gentle way that the granule membrane is still intact. Unruptured and ruptured cells were fixed in 2% paraformaldehyde and 0.075% glutaraldehyde, dehydrated in ethanol. For TEM (transmission electron microscopy) cells were embedded in Lowicryl K4M at -35°C and for SEM (scanning electron microscopy) they were placed on copper blocks, critical point dried and coated. For immunoelectron microscopy ultrathin sections were incubated with monoclonal anti-actin and colloidal gold labelled IgM. Ruptured cells were also placed on cover glasses, prefixed, and incubated with anti-actin and colloidal gold labelled IgM.

Actin filaments in two spermatozoa of crustacea decapoda

1990 ◽  
Vol 48 (3) ◽  
pp. 70-71
Author(s):  
E. Dupré ◽  
G. Schatten
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Low Voltage ◽  
Active Role ◽  
Main Body ◽  
Robinson Crusoe ◽  
Decapod Crustaceans ◽  
Actual Participation ◽  
Voltage Scanning ◽  
Scanning Electron

Sperm of decapod crustaceans are formed by a round or cup-shaped body, a complex acrosome and one a few appendages emerging from the main body. Although this sperm does not have motility, it has some components of the cytoskeleton like microtubules, which are found inside the appendages. Actin filaments have been found in the spike of penaeidae sperms. The actual participation of the crustacean decapod sperm cytoskeleton during fertilization is not well understood. Actin is supposed to play an active role in drawing the penaeidae shrimp sperm closer to the egg after bending of the spike. The present study was aimed at the localization of actin filaments in sperm of the Robinson Crusoe island lobster, Jasus frontalis and in the crayfish Orconectes propincus, by fluorescent probes and low voltage scanning electron microscopy.

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