scholarly journals Banding and polarity of actin filaments in interphase and cleaving cells.

1980 ◽  
Vol 86 (2) ◽  
pp. 568-575 ◽  
Author(s):  
J M Sanger ◽  
J W Sanger
Keyword(s):  
Electron Microscopy ◽  
Tannic Acid ◽  
Actin Filaments ◽  
Cell Junctions ◽  
Stress Fibers ◽  
Cleavage Furrow ◽  
Heavy Meromyosin ◽  
Glutaraldehyde Fixation ◽  
Microfilament Bundles ◽  
Uniform Polarity

Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments.

scholarly journals Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid.

1979 ◽  
Vol 80 (3) ◽  
pp. 759-766 ◽  
Author(s):  
R D Goldman ◽  
B Chojnacki ◽  
M J Yerna
Keyword(s):  
Electron Microscope ◽  
Electron Density ◽  
Tannic Acid ◽  
Osmium Tetroxide ◽  
Stress Fibers ◽  
Supramolecular Organization ◽  
Glutaraldehyde Fixation ◽  
Microfilament Bundles ◽  
Electron Density Shifts

After standard glutaraldehyde-osmium tetroxide fixation procedures, the majority of microfilament bundles in BHK-21 cells exhibit relatively uniform electron density along their long axes. The inclusion of tannic acid in the glutaraldehyde fixation solution results in obvious electron density shifts along the majority of microfilament bundles. Striated patterens are frequently observed which consist of regularly spaced electron dense (D) and electron lucid (L) bands. A striated pattern is also observed along many BHK-21 stress fibers after processing for indirect immunofluorescence utilizing BHK-21 myosin antiserum. A direct correlation of these periodicities seen by light and electron microscope techniques is impossible at the present time. However, comparative measurements indicate that the overall patterns seen in the immunofluorescence and electron microscope preparations are similar. The ultrastructural results provide an initial clue for the ultimate determination of the supramolecular organization of contracile proteins other than actin within the microfilament bundles of non-muscle cells.

scholarly journals FILAMENT ULTRASTRUCTURE AND ORGANIZATION IN VERTEBRATE SMOOTH MUSCLE

1967 ◽  
Vol 35 (2) ◽  
pp. 303-321 ◽  
Author(s):  
Bernard J. Panner ◽  
Carl R. Honig
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Actin Filaments ◽  
Heavy Meromyosin ◽  
Dense Bodies ◽  
Contraction Mechanism ◽  
Dark Staining ◽  
Technical Factors ◽  
Low Ionic Strength

Using a variety of preparative techniques for electron microscopy, we have obtained evidence for the disposition of actin and myosin in vertebrate smooth muscle. All longitudinal myofilaments seen in sections appear to be actin. Previous reports of two types of longitudinal filaments in sections are accounted for by technical factors, and by differentiated areas of opacity along individual filaments. Dense bodies with actin emerging from both ends have been identified in homogenates, and resemble Z discs from skeletal muscle (Huxley, 1963). In sections, short, dark-staining lateral filaments 15–25 A in diameter link adjacent actin filaments within dense bodies and in membrane dense pataches. They appear homologous with Z-disc filaments. Similar lateral filaments connect actin to plasma membrane. Dense bodies and dense patches, therefore, are attachment points and denote units analogous to sarcomeres. In glycerinated, methacrylate-embedded sections, lateral processes different in length and staining characteristics from lateral filaments in dense bodies exist at intervals along actin filaments. These processes are about 30 A wide and resemble heavy meromyosin from skeletal muscle. They also resemble heads of whole molecules of myosin in negatively stained material from gizzard homogenates. Intact single myosin molecules and dimers have been found, both free and attached to actin, even in media of very low ionic strength. Myosin can, therefore, exist in relatively disaggregated form. Models of the contraction mechanism of smooth muscle are proposed. The unique features are: (1) Myosin exists as small functional units. (2) Movement occurs by interdigitation and sliding of actin filaments.

Negative Staining of F-Actin in situ with Tannic Acid

1975 ◽  
Vol 33 ◽  
pp. 628-629
Author(s):  
James R. LaFountain ◽  
Herbert R. Thomas
Keyword(s):  
Electron Microscopy ◽  
Tannic Acid ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Vastus Lateralis ◽  
Muscle Cells ◽  
Cacodylate Buffer ◽  
Spindle Apparatus ◽  
Ultrastructural Findings

Since the introduction of tannic acid as an additive in glutaraldehyde fixatives for electron microscopy, there have been numerous reports of ultrastructural findings that were not detectable after fixation without tannic acid. We have used tannic acid in studies on the spindle apparatus in insect spermatocytes to show that microtubules of the spindle are composed of 13 protofilaments. Tannic acid accumulates at the periphery of subunits of microtubules and with osmium stains those regions, leaving the subunits unstained. Hence, the subunits appear negatively stained.We have found that in addition to microtubules, other filamentous structures in cells appear negatively stained after fixation with glutaraldehyde-tannic acid. The most noteable are actin filaments in muscle cells. We report here results obtained from mouse striated muscle.Segments of gastrocnemius and vastus lateralis muscles are dissected in 3% glutaraldehyde in 0.1 M cacodylate buffer and subsequently fixed for 1 hr in the above fixative containing tannic acid (Mallinckrodt) in concentrations of 4 and 8%. Both concentrations gave similar results.

Fixation and conductive staining by tannin-RuO4 for transmission and scanning electron microscopy

1990 ◽  
Vol 48 (3) ◽  
pp. 24-25
Author(s):  
Gen Takahashi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Thin Section ◽  
Tannic Acid ◽  
Osmium Tetroxide ◽  
Glutaraldehyde Fixation ◽  
Ruthenium Tetroxide ◽  
Cellular Ultrastructure ◽  
Scanning Electron

Ruthenium tetroxide(RuO4) was first used as a stain in histology by Ranvier in 1887. Its use in TEM as a fixative or stain was reported by Gaylarde et al. However,the preservation of the cellular ultrastructure was very poor after RuO4 fixation alone, because RuO4 is a far more vigorous oxidant than osmium tetroxide. Peltari et al reported that a preceeding glutaraldehyde fixation helped to stabilize the ultrastructure, although the penetration of RuO4 into the tissue was poor.In the present study, RuO4 has been found to be a useful fixative and staining reagent with the prerequisite of using RuO4 as a postfixative after prefixation with tannic acid (TA)-glutaraldehyde(GL) for thin-section TEM(TA-RuO4 method) or after preceeding osmication followed by TA mordanting for non-coating SEM(OsO4-TA-RuO4 method).TA-RuO4 method for thin-section TEM

scholarly journals Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation.

1983 ◽  
Vol 96 (1) ◽  
pp. 51-62 ◽  
Author(s):  
P Maupin ◽  
T D Pollard
Keyword(s):  
Tannic Acid ◽  
Hela Cells ◽  
Actin Filaments ◽  
Amorphous Material ◽  
Stress Fibers ◽  
Cell Cortex ◽  
Intact Cells ◽  
Cytoplasmic Matrix ◽  
Cytoplasmic Structures ◽  
Coated Membrane

Fixation of HeLa cells with a mixture of 100 mM glutaraldehyde, 2 mg/ml tannic acid and 0.5 mg/ml saponin allows the tannic acid to penetrate intact cells without disruption of membranes or extraction of the cytoplasmic matrix. After subsequent treatment with OsO4 cytoplasmic structures are stained so densely that fine details are visible even in very thin (dark gray) sections. Actin filaments are protected from disruption by OsO4 so that straight, densely stained filaments are seen in the cell cortex, filopodia, ruffling membranes, and stress fibers. Stress fibers also have 15-18-nm densities similar in appearance to myosin filaments. Tannic acid staining reveals that the coats of coated vesicles, pits, and plaques have a 12-nm layer of amorphous material between the membrane and the clathrin basketwork. HeLa cells have very large clathrin-coated membrane plaques on the basal surface. These coated membrane plaques appear to be a previously unrecognized site of cell-substrate adhesion.

scholarly journals Calcium-regulated cooperative binding of the microvillar 110K-calmodulin complex to F-actin: formation of decorated filaments.

1987 ◽  
Vol 105 (1) ◽  
pp. 325-333 ◽  
Author(s):  
L M Coluccio ◽  
A Bretscher
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Protein Concentration ◽  
High Yield ◽  
Cooperative Binding ◽  
Compelling Evidence ◽  
Calmodulin Antagonists ◽  
Heavy Meromyosin ◽  
The Core ◽  
Purification Scheme

The 110K-calmodulin complex of intestinal microvilli is believed to be the link between the actin filaments comprising the core bundle and the surrounding cell membrane. Although not the first study describing a purification scheme for the 110K-calmodulin complex, a procedure for the isolation of stable 110K-calmodulin complex both pure and in high yield is presented; moreover, isolation is without loss of the associated calmodulin molecules since a previously determined ratio in isolated microvillar cytoskeletons of calmodulin to 110-kD polypeptide of 3.3:1 is preserved. We have found that removal of calmodulin from the complex by the calmodulin antagonists W7 or W13 results in precipitation of the 110-kD polypeptide with calmodulin remaining in solution. The interaction of 110K-calmodulin with beef skeletal muscle F-actin has been examined. Cosedimentation assays of 110K-calmodulin samples incubated with F-actin show the amount of 110K-calmodulin associating with F-actin to be ATP, calcium, and protein concentration dependent; however, relatively salt independent. In calcium, approximately 30% of the calmodulin remains in the supernatant rather than cosedimenting with the 110-kD polypeptide and actin. Electron microscopy of actin filaments after incubation with 110K-calmodulin in either calcium- or EGTA-containing buffers show polarized filaments often laterally associated. Each individual actin filament is seen to exhibit an arrowhead appearance characteristic of actin filaments after their incubation with myosin fragments, heavy meromyosin and subfragment 1. In some cases projections having a 33-nm periodicity are observed. This formation of periodically spaced projections on actin filaments provides further compelling evidence that the 110K-calmodulin complex is the bridge between actin and the microvillar membrane.

scholarly journals Evidence for actin involvement in cardiac Z-lines and Z-line analogues.

1983 ◽  
Vol 96 (2) ◽  
pp. 435-442 ◽  
Author(s):  
M Yamaguchi ◽  
R M Robson ◽  
M H Stromer
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Muscle Fibers ◽  
Opposite Polarity ◽  
Dense Material ◽  
Heavy Meromyosin ◽  
Thin Filaments ◽  
Electron Dense Material ◽  
Before And After

Canine and feline cardiac Z-lines and Z-rods were examined by electron microscopy before and after digestion of muscle fibers with Ca2+-activated protease (CAF). Removal by CAF of electron-dense material which covers Z-lines and Z-rods exposed interdigitating longitudinal filaments (6-7 nm in diameter) apparently continuous with thin filaments of the respective I-bands. The newly exposed longitudinal filaments of CAF-treated Z-lines and of CAF-treated Z-rods bound heavy meromyosin and therefore are actin. The width of Z-lines and length of Z-rods are determined by the amount of overlap of actin filaments of opposite polarity. The oblique filaments in Z-lines and Z-rods are responsible for the perpendicular periodicity of Z-lines and Z-rods, and are attributed to alpha-actinin.

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.

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.

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.

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