scholarly journals Actin filament destruction by osmium tetroxide

1978 ◽  
Vol 77 (3) ◽  
pp. 837-852 ◽  
Author(s):  
P Maupin-Szamier ◽  
TD Pollard
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Sodium Phosphate ◽  
Osmium Tetroxide ◽  
Time Span ◽  
Cross Linking ◽  
Muscle Actin ◽  
Viscosity Change ◽  
Sodium Phosphate Buffer ◽  
Sulfur Containing

We have studied the destruction of purified muscle actin filaments by osmium tetroxide (OsO4) to develop methods to preserve actin filaments during preparation for electron microscopy. Actin filaments are fragmented during exposure to OsO4. This causes the viscosity of solutions of actin filaments to decrease, ultimately to zero, and provides a convenient quantitative assay to analyze the reaction. The rate of filament destruction is determined by the OsO4 concentration, temperature, buffer type and concentration, and pH. Filament destruction is minimized by treatment with a low concentration of OsO4 in sodium phosphate buffer, pH 6.0, at 0 degrees C. Under these conditions, the viscosity of actin filament solutions is stable and actin filaments retain their straight, unbranched structure, even after dehydration and embedding. Under more severe conditions, the straight actin filaments are converted into what look like the microfilament networks commonly observed in cells fixed with OsO4. Destruction of actin filaments can be inhibited by binding tropomyosin to the actin. Cross-linking the actin molecules within a filament with glutaraldehyde does not prevent their destruction by OsO4. The viscosity decrease requires the continued presence of free OsO4. During the time of the viscosity change, OsO4 is reduced and the sulfur-containing amino acids of actin are oxidized, but little of the osmium is bound to the actin. Over a much longer time span, the actin molecules are split into discrete peptides.

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.

scholarly journals The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

2016 ◽  
Vol 27 (11) ◽  
pp. 1821-1833 ◽  
Author(s):  
Yujie Li ◽  
Jenna R. Christensen ◽  
Kaitlin E. Homa ◽  
Glen M. Hocky ◽  
Alice Fok ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Actin Filament ◽  
Actin Filaments ◽  
Biochemical Properties ◽  
Ring Formation ◽  
Cross Linking ◽  
Contractile Ring ◽  
Mixed Polarity ◽  
Dividing Cells

The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.

scholarly journals Bundling of actin filaments by alpha-actinin depends on its molecular length.

1990 ◽  
Vol 110 (6) ◽  
pp. 2013-2024 ◽  
Author(s):  
R K Meyer ◽  
U Aebi
Keyword(s):  
Smooth Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Molar Ratio ◽  
Cross Linking ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Chicken Gizzard ◽  
Molecular Length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

scholarly journals Isoforms of α-Actinin from Cardiac, Smooth, and Skeletal Muscle Form Polar Arrays of Actin Filaments

2000 ◽  
Vol 149 (3) ◽  
pp. 635-646 ◽  
Author(s):  
Kenneth A. Taylor ◽  
Dianne W. Taylor ◽  
Fred Schachat
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Polar Filament ◽  
Relative Orientation ◽  
Lipid Monolayer ◽  
Cross Linking ◽  
Internal Diameter ◽  
Vertebrate Muscle ◽  
Filament Bundles ◽  
Positively Charged

We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by α-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using α-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4–0.7 μm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that α-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by α-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of α-actinin.

Coagulation factor Va is an actin filament binding and cross-linking protein

1995 ◽  
Vol 73 (1-2) ◽  
pp. 105-112 ◽  
Author(s):  
Emilia Furmaniak-Kazmierczak ◽  
Michael E. Nesheim ◽  
Graham P. Côté
Keyword(s):  
Actin Filament ◽  
Light Chain ◽  
Actin Filaments ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Actin Binding ◽  
Cross Linking ◽  
Factor V ◽  
Proteolytic Activation ◽  
Factor Va

Bovine coagulation cofactor factor Va is shown to bind to filaments of skeletal muscle actin with a dissociation constant of 40–50 nM in the presence of 50 mM NaCl. At saturation, approximately one molecule of factor Va was bound for every two actin molecules. The binding of factor Va to F-actin was inhibited by increasing ionic strength, being approximately 20-fold weaker at 150 mM NaCl. Addition of factor Va dramatically increased both the low-speed sedimentation and the low-shear viscosity of actin filament solutions, indicating that factor Va cross-links actin filaments. Factor Va also bound to actin filaments saturated with myosin. The isolated 74-kilodalton light chain of factor Va displayed actin binding and cross-linking properties indistinguishable from those of intact factor Va. The procofactor factor V bound weakly to F-actin, indicating that proteolytic activation is required to uncover the actin binding sites within the light chain domain. Actin filaments had only a slight inhibitory effect on the prothombinase activity of the factor Va – factor Xa – phospholipid complex. Since high concentrations of actin filaments can be exposed to the circulation when cells are damaged, the interaction of factor Va with actin may be of physiological relevance.Key words: blood coagulation, factor V, actin.

scholarly journals Profilin and formin constitute a pacemaker system for robust actin filament growth

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Johanna Funk ◽  
Felipe Merino ◽  
Larisa Venkova ◽  
Lina Heydenreich ◽  
Jan Kierfeld ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Growth Rates ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Biological Processes ◽  
Muscle Actin ◽  
Cell Morphogenesis ◽  
Actin Networks ◽  
In Cells

The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.

Fine Structure of Spermatogenesis in the Snail Tarebia Granifera (Melaniidae)

1977 ◽  
Vol 35 ◽  
pp. 620-621
Author(s):  
Ronald W. Scates ◽  
Robert V. Blystone
Keyword(s):  
Electron Microscopy ◽  
Sodium Phosphate ◽  
Osmium Tetroxide ◽  
Reproductive Tract ◽  
San Antonio ◽  
Snail Population ◽  
Sodium Phosphate Buffer ◽  
Apomictic Parthenogenesis ◽  
Comprehensive Study ◽  
Tarebia Granifera

Jacob (1), citing chromosomal studies, emphasized the extreme rarity, or possible non-existence of viable males in observed Indian populations of Melanoides tuberculatus, of the same family as Tarebia granifera. The snail population, almost totally female in chromosomal count, were presumed to reproduce parthenogenetically; no sexual reproduction was in evidence (no sperm were ever reported observed). Similar findings were reported by Pace (2) in his comprehensive study of populations of T. granifera on the island of Taiwan. Pace, correlating his own findings with those of Jacob, suggested that T. granifera reproduce by apomictic parthenogenesis.A population of T. granifera is found in Brackenridge Park, San Antonio, Texas. The reproductive tract of randomly selected snails was prepared for electron microscopy. The tissue was fixed in 3% glutaraldehyde in 0.1 M sodium phosphate buffer, postfixed in buffered 1% Osmium tetroxide, dehydrated, and embedded in Spurr embedding media.

scholarly journals Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks.

1981 ◽  
Vol 91 (3) ◽  
pp. 695-705 ◽  
Author(s):  
J V Small
Keyword(s):  
Electron Microscopy ◽  
Actin Filament ◽  
Actin Filaments ◽  
Osmium Tetroxide ◽  
Cultured Cells ◽  
Leading Edge ◽  
Cultured Fibroblasts ◽  
Cell Biol ◽  
Filament Bundles

The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X-100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed.

scholarly journals Under physiological conditions actin disassembles slowly from the nonpreferred end of an actin filament.

1983 ◽  
Vol 97 (5) ◽  
pp. 1629-1634 ◽  
Author(s):  
L M Coluccio ◽  
L G Tilney
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Actin Filament ◽  
Salt Concentration ◽  
Actin Filaments ◽  
Average Length ◽  
Muscle Actin ◽  
Subunit Exchange ◽  
Physiological Conditions ◽  
Cellular Factors

Incubation of the isolated acrosomal bundles of Limulus sperm with skeletal muscle actin results in assembly of actin onto both ends of the bundles. Because of the taper of these cross-linked bundles of actin filaments, one can distinguish directly the preferred end for assembly from the nonpreferred end. Loss of growth with time from the nonpreferred end was directly assessed by electron microscopy and found to be dependent upon salt concentration. Under physiological conditions (100 mM KCl, 1 mM MgCl2) and excess ATP (0.5 mM), depolymerization of the newly assembled actin filaments at the nonpreferred end over an 8-h period was 0.024 micron/h. Thus, even after 8 h, 63% of the bundles retained significant growth on their nonpreferred ends, the average length being 0.21 micron +/- 0.04. However, in the presence of 1.2 mM CaCl2, disassembly of actin monomers from the nonpreferred end increased substantially. By 8 h, only 7% of the bundles retained any actin growth on the nonpreferred ends, and the depolymerization rate off the nonpreferred end was 0.087 micron/h. From these results we conclude that, in the absence of other cellular factors, disassembly of actin subunits from actin filaments (subunit exchange) is too slow to influence most of the motile events that occur in cells. We discuss how this relates to treadmilling.

scholarly journals Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

1998 ◽  
Vol 9 (4) ◽  
pp. 841-852 ◽  
Author(s):  
R. Dyche Mullins ◽  
Joseph F. Kelleher ◽  
James Xu ◽  
Thomas D. Pollard
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Analytical Ultracentrifugation ◽  
Acanthamoeba Castellanii ◽  
Leading Edge ◽  
Actin Binding ◽  
Cross Linking ◽  
Motile Cells ◽  
Mechanism Of Interaction ◽  
Cross Links

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motileAcanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.

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