Actin Dimer Research Articles

Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane.

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin-reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.

Membrane protein organization in ATP-depleted and irreversibly sickled red cells.

AbstractIt has been proposed that the spectrin‐actin submembrane network participates in control of red cell shape and deformability. We have examined ATP‐ and calcium‐dependent changes in organization of spectrin in the membrane employing cross‐linking of the nearest membrane protein neighbors by spontaneous or catalyzed (CuSO4, O‐phenanthroline) intermolecular disulfide couplings and two‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis.Cross‐linking of fresh red cells resulted in the formation of spectrin and actin dimers and tetramers. ATP‐depleted red cells differed from fresh cells in the presence of an additional reducible polymer of MW &gt; 1 × 106 selectively enriched in spectrin. This polymer formed spontaneously when red cells were depleted of ATP under aerobic conditions. After anaerobic ATP depletion, the polymer formed in ghosts after cross‐linking by catalytic oxidation. Polymerization was prevented by maintenance of ATP and coincided with an ATP‐dependent discocyte‐echinocyte transformation. This suggests that, in ATP‐depleted red cells, spectrin is rearranged to establish closer contacts, and that this may contribute to the discocyte‐echinocyte transformation.The introduction of greater than 0.5 mM Ca++ into ghosts by inclusion in hemolysis buffer or into fresh red cells (but not ATP‐depleted red cells) by treatment with ionophore A23187 spontaneously produced a nonreducible polymer which others have attributed to transamidative cross‐linking of spectrin, band 3, and other proteins. Spontaneous formation of both polymer types (reducible in aerobically ATP‐depleted red cells and nonreducible in fresh, Ca++ enriched red cells) resulted in stabilization (“autocatalytic fixation”) of spheroechinocytic shape.Irreversibly sickled cells, which have increased calcium and decreased ATP, and exhibit a permanent membrane deformation, failed to form any of the above polymers. This suggests that in contrast to normal cells depleted of ATP in vitro, fixation of ISC shape in vivo is not related to Ca‐ and ATP‐dependent membrane protein polymerization. However, ISCs had an increased propensity to form the reducible, spectrin‐rich polymer during a subsequent metabolic depletion in vitro. This was associated with transformation of ISCs into spheroechinocytes. Similar echinocytic ISCs were found to constitute 5–10% of the densest fractions of freshly separated ISCs. ISCs then exhibit sphero‐echniocyte transformation, both in vitro and in vivo. We propose that this is due to spectrin reorganization that presumably results from the progressively increasing calcium and decreasing ATP of ISCs.These data provide evidence of altered spectrin organization in membranes of ATP‐depleted, calcium‐enriched red cells in vitro and in vivo.

Freezing of actin. Reversible oxidation of a sulfhydryl group and structural change.

It was found that the essential change in actin (whether G- or F-actin) on freeze-thawing was the specific oxidation of one of five sulfhydryl (SH) groups, i.e. the SH group of Cys 373 in the amino acid sequence. Oxidized SH groups formed an inter-molecular disulfide (SS) bond to yield an actin dimer. F-actin, subjected to freeze-thawing (or F-actin obtained by the transformation of once frozen G-actin which is essentially a dimer), has anomalous physico-chemical properties and a different conformation from normal F-actin, as determined by optical and electron microscopic observations, as well as high steady ATP-splitting activity in the presence of Mg2+. However, it was found that those peculiarities disappeared and normal actin was reformed on reducing the oxidized SH group with dithiothreitol (DTT). It was also found that the normal characteristics of actin were preserved for more than four months on freezing in the presence of a sufficient amount of DTT.

The interaction of actin, myosin and adenosine triphosphate

1. 1. Purified actin has been coupled with a fluorescent dye, 1-dimethylaminonaphthalene-5-sulphonyl chloride, which enables the molecular state of aggregation of F-actin and of F-actomyosin to be investigated by the fluorestscence-polarisation technique of G. Weber. The coupling does not modify the physico-chemical properties of actin with respect to “polymerisability” and ability to form actomyosin. 2. 2. The kinetic units in F-actin are those of the actin dimers of particle weight 140,000 and rotational relaxation time ∼32·10 −8 sec. There is no evidence for the existence in solution of polymerised fibrils such as are seen after drying nor of the primary large aggregates (“ovoids”) which might act as their precursors. 3. 3. There appears to be no complex formation between myosin and F-actin. In F-actomyosin, the actin partner exists in the monomeric form. 4. 4. Alongside the macroscopic changes in viscosity, flow birefringence, light scattering and sedimentation, ATP appear to cause instantaneous re-dimerisation of the actin partner. 5. 5. These observations and the published results of other investigators using different techniques are discussed in relation to the alternative views of polymerisation as against the formation of dynamic structures in solution.