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 Mechanism of interaction of Dictyostelium severin with actin filaments.

1982 ◽  
Vol 95 (3) ◽  
pp. 711-719 ◽  
Author(s):  
K Yamamoto ◽  
J D Pardee ◽  
J Reidler ◽  
L Stryer ◽  
J A Spudich
Keyword(s):  
Energy Transfer ◽  
Actin Filaments ◽  
Exchange Process ◽  
Molar Ratio ◽  
Fluorescence Energy Transfer ◽  
Dependent Manner ◽  
Emission Anisotropy ◽  
Subunit Exchange ◽  
Fluorescence Energy

Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+ -dependent manner, was purified 500-fold to greater than 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29 A and consists of a single polypeptide chain. It contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluoresence-labeled severin shows that this protein changes its conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 approximately equal to 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 approximately equal to 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin: severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo.

scholarly journals Detection of actin assembly by fluorescence energy transfer.

1981 ◽  
Vol 89 (2) ◽  
pp. 362-367 ◽  
Author(s):  
D L Taylor ◽  
J Reidler ◽  
J A Spudich ◽  
L Stryer
Keyword(s):  
Energy Transfer ◽  
Actin Filaments ◽  
Radial Coordinate ◽  
Fluorescence Energy Transfer ◽  
Actin Assembly ◽  
Intact Cells ◽  
Donor And Acceptor ◽  
Fluorescence Energy ◽  
Energy Acceptor

Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor-labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole fraction of acceptor-labeled actin showed that the radial coordinate of the label at cysteine 373 is approximately 35 A, which means that this site is located near the outer surface of the filament. The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.

Actin and myosin: control of filament assembly

1982 ◽  
Vol 299 (1095) ◽  
pp. 247-261 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Thick Filament ◽  
Light Chains ◽  
Fluorescence Energy Transfer ◽  
Dependent Manner ◽  
Filament Formation ◽  
Tail Region ◽  
Filament Assembly ◽  
Fluorescence Energy

Actin filaments, assembled from highly purified actin from either skeletal muscle or Dictyostelium amoebae, are very stable under physiological ionic conditions. A small and limited amount of exchange of actin filament subunits for unpolymerized actin or subunits in other filaments has been measured by three techniques: fluorescence energy transfer, incorporation of 35 S-labelled actin monomers into unlabelled actin filaments, and exchange of [ 14 C]ATP with filament-bound ADP. A 40 kDa protein purified from amoebae destabilizes these otherwise stable filaments in a Ca 2+ -dependent manner. Myosin purified from Dictyostelium amoebae is phosphorylated both in the tail region of the heavy chain and in one of the light chains. Phosphorylation appears to regulate myosin thick-filament formation.

scholarly journals Changes in fluorescence energy transfer between sulfhydryl fluorescent residues during ouabain sensitive Na+, K+-ATP hydrolysis.

1987 ◽  
Vol 44 (3) ◽  
pp. 311-321
Author(s):  
Masahiko SAKURAYA ◽  
Kazuya TANIGUCHI ◽  
Kuniaki SUZUKI ◽  
Akinobu KUDO ◽  
Shinji NAKAMURA ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Atp Hydrolysis ◽  
Fluorescence Energy Transfer ◽  
Fluorescence Energy

The capactins, a class of proteins that cap the ends of actin filaments

1982 ◽  
Vol 299 (1095) ◽  
pp. 263-273 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Preliminary Evidence ◽  
The Other ◽  
Cellular Regulation ◽  
Filament Assembly ◽  
Filament Bundles

A number of proteins that bind specifically to the barbed ends of actin filaments in a cytochalasin-like manner have been purified to various degrees from a variety of muscle and non-muscle cells and tissues. Preliminary evidence also indicates that proteins that interact with the pointed ends of filaments are present in skeletal muscle. Because of their ability to cap one or the other end of an actin filament, we have designated this class of proteins as the ‘capactins’. On the basis of their effect on actin filament assembly and interaction in vitro , we propose that the capactins play important roles in cellular regulation of actin-based cytoskeletal and contractile functions. Our finding that the disappearance of actin filament bundles in virally transformed fibroblasts can be correlated with an increase in capactin activity in the extracts of these cells is consistent with this hypothesis.

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.

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