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 Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Muscle Actin ◽  
Hydrophobic Cavity ◽  
Cryo Electron Microscopy ◽  
Kinetics Of

AbstractSince the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.

scholarly journals Actin filaments undergo limited subunit exchange in physiological salt conditions.

1982 ◽  
Vol 94 (2) ◽  
pp. 316-324 ◽  
Author(s):  
J D Pardee ◽  
P A Simpson ◽  
L Stryer ◽  
J A Spudich
Keyword(s):  
Skeletal Muscle ◽  
Energy Transfer ◽  
Actin Filament ◽  
Actin Filaments ◽  
Atp Hydrolysis ◽  
Fluorescence Energy Transfer ◽  
Subunit Exchange ◽  
Filament Assembly ◽  
Fluorescence Energy ◽  
Energy Acceptor

The exchange of actin filament subunits for unpolymerized actin or for subunits in other filaments has been quantitated by three experimental techniques: fluorescence energy transfer, incorporation of 35S-labeled actin monomers into unlabeled actin filaments, and exchange of [14C]ATP with filament-bound ADP. In the fluorescence energy transfer experiments, actin labeled with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (IAENS) served as the fluorescent energy donor, and actin labeled with either fluorescein-5-isothiocyanate (FITC) or fluorescein-5-maleimide (FM) served as the energy acceptor. Fluorescent-labeled actins from Dictyostelium amoebae and rabbit skeletal muscle were very similar to their unlabeled counterparts with respect to critical actin concentration for filament assembly, assembly rate, ATP hydrolysis upon assembly, and steady-state ATPase. As evidenced by two different types of fluorescence energy transfer experiments, less than 5% of the actin filament subunits exchanged under a variety of buffer conditions at actin concentrations greater than 0.5 mg/ml. At all actin concentrations limited exchange to a plateau level occurred with a half-time of about 20 min. Nearly identical results were obtained when exchange was quantitated by incorporation of 35S-labeled Dictyostelium actin monomers into unlabeled muscle actin or Dictyostelium actin filaments. Furthermore, the proportion of filament-bound ADP which exchanged with [14C]-ATP was nearly the same as actin subunit exchange measured by fluorescence energy transfer and 35S-labeled actin incorporation. These experiments demonstrate that under approximately physiologic ionic conditions only a small percentage of subunits in highly purified skeletal muscle or Dictyostelium F-actin participate in exchange.

scholarly journals Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs.

1979 ◽  
Vol 82 (1) ◽  
pp. 212-226 ◽  
Author(s):  
A Spudich ◽  
J A Spudich
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Actin Filaments ◽  
Cell Types ◽  
Optical Diffraction ◽  
Muscle Actin ◽  
Sea Urchin Eggs ◽  
Inner Surface ◽  
Low Ionic Strength ◽  
Helical Parameters

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.

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.

Polymorphism of Actin Paracrystals

1981 ◽  
Vol 39 ◽  
pp. 420-421
Author(s):  
W.E. Fowler ◽  
U. Aebi
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Muscle Cells ◽  
New Method ◽  
Structural Studies ◽  
Muscle Actin ◽  
Structural Detail ◽  
Naturally Occurring ◽  
Synthetic Filament

In the muscle sarcomere and in certain specialized non-muscle cells actin filaments are organized in bundles or paracrystalline arrays. Structural studies of these naturally occurring filament arrays have been limited to about 3nm resolution, mainly due to inherent disorder of the specimen and/or difficulties with the preparation of these arrays for EM. Skeletal muscle G-actin can be induced to form synthetic filament paracrystals upon addition of non-physiological concentrations of Mg++ (e.g. 50mM) . The structural resolution obtained with these synthetic paracrystals has been of the same order (about 3nm) as that encountered with the naturally occurring filament arrays. Using a new method of induction, we have obtained synthetic paracrystals with two non-muscle actins which reveal structural detail to almost 2nm resolution (Figs. 1,2,3). While the same types of paracrystals were observed with Physarum and Acanthamoeba actin, skeletal muscle actin displayed a different polymorphism under identical conditions.

Nonmuscle tropomyosin-4 requires coexpression with other low molecular weight isoforms for binding to thin filaments in cardiomyocytes

1999 ◽  
Vol 112 (3) ◽  
pp. 371-380 ◽  
Author(s):  
D.M. Helfman ◽  
C. Berthier ◽  
J. Grossman ◽  
M. Leu ◽  
E. Ehler ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Actin Filament ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Synergistic Effects ◽  
Neonatal Rat ◽  
Low Molecular Weight ◽  
Rat Cardiomyocytes ◽  
Filament Dynamics

Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms are generated via a complex pattern of alternative RNA splicing and alternative promoters. The functional significance of this isoform diversity is largely unknown and it remains to be determined whether specific isoforms are required for assembly and integration into distinct actin-containing structures. The ability of nonmuscle (TM-1, -2, -3, -4, -5(NM1), -5a or -5b) and striated muscle (skeletal muscle (α)-TM) isoforms to incorporate into actin filaments of neonatal rat cardiomyocytes (NRCs) was studied using expression plasmids containing TM-fusions with GFP (green fluorescent protein) as well as with VSV- or HA-epitope tags. All isoforms, except of fibroblast TM-4, were able to incorporate into the I-band of NRCs. When TM-4 was co-transfected with other low molecular weight (LMW) isoforms of TM (TM-5, TM-5a and TM-5b), it was able to incorporate into sarcomeres of NRCs. This result was not obtained when TM-4 was co-transfected with high molecular weight (HMW) TMs (TM-1, TM-2 or skeletal muscle (α)-TM). These data demonstrate that the ability of TM-4 to bind to actin filaments can be specifically influenced by its interaction with other LMW TM isoforms. In addition, cells that incorporated the muscle or nonmuscle GFP-TMs into their sarcomeres continued to beat and exhibited sarcomeric contraction. These studies provide the first in vivo demonstration of synergistic effects between TM isoforms for binding to actin filaments. These results have important implications in understanding actin filament dynamics in nonmuscle cell systems, especially during development and in transformed cells, where alterations in the ratio of different LMW isoforms might lead to changes in their interactions with actin filaments. Furthermore, these studies demonstrate that GFP-TM can be used to study thin-filament dynamics in muscle cells and actin filament dynamics in nonmuscle 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.

Factors affecting movement of F-actin filaments propelled by skeletal muscle heavy meromyosin

1992 ◽  
Vol 262 (3) ◽  
pp. C714-C723 ◽  
Author(s):  
E. Homsher ◽  
F. Wang ◽  
J. R. Sellers
Keyword(s):  
Skeletal Muscle ◽  
Ionic Strength ◽  
Actin Filament ◽  
Molecular Motors ◽  
Actin Filaments ◽  
Heavy Meromyosin ◽  
Shortening Velocity ◽  
Weakly Bound ◽  
Factors Affecting ◽  
Cross Bridges

The measurement of fluorescent-labeled actin filament movement driven by mechanoenzymes (e.g., myosin) is an important methodology for the study of molecular motors. It is assumed that the filament velocity (Vf) is analogous to the unloaded shortening velocity (Vu) seen in muscle fibers. Methods are described to reproducibly quantitate the movement of these filaments and to select uniformly moving filaments and specify their Vf. Use of these techniques allowed comparison of Vf to literature values for Vu with regard to [ATP], [ADP], [Pi], pH, ionic strength (10-150 mM), and temperature (15-30 degrees C). Vf and Vu are quantitatively similar with respect to the effects of substrate and product concentrations and temperatures greater than 20 degrees C. However, Vf is more sensitive to decreases in pH and temperatures less than 20 degrees C than Vu. At ionic strengths of 50-150 mM, Vf and Vu exhibit similar ionic strength dependencies (decreasing with ionic strength). At ionic strengths less than 50 mM, Vf is markedly reduced. Results of experiments using adenosine 5'-O-(3-thiotriphosphate) suggest that increasing the number of weakly bound cross bridges does not seriously affect Vf. Thus, although Vf is a good analogue for Vu under certain conditions (elevated ionic strength and temperatures greater than 20 degrees C), under others it is not. The results of motility assays must be cautiously interpreted.

scholarly journals Antigenic probes locate a serum-gelsolin-interaction site on the C-terminal part of actin

1987 ◽  
Vol 248 (2) ◽  
pp. 359-364 ◽  
Author(s):  
M Boyer ◽  
J Feinberg ◽  
H K Hue ◽  
J P Capony ◽  
Y Benyamin ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filament ◽  
Muscle Actin ◽  
Terminal Part ◽  
Interaction Site ◽  
Specific Antibodies

The implication of part of the C-terminal of actin (within the 285-375 sequence) in the interaction of serum gelsolin was investigated by the use of specific antibodies. These antibodies were directed against two or three discrete epitopes, one of which was specific for skeletal-muscle actin. Some of these epitopes were found to be near the serum gelsolin-actin interface. Thus it can be assumed that part of the C-terminal of actin is exposed at the barbed end of the actin filament. The interaction between tropomyosin and actin was also studied.

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