Pointed Ends Of Filaments Research Articles

Difference in polymerization and steady-state dynamics of free and gelsolin-capped filaments formed by α- and β-isoactins

The polymerization of scallop β-like actin is significantly slower than that of skeletal muscle α-actin. To reveal which steps of polymerization contribute to this difference, we estimated the efficiency of nucleation of the two actins, the rates of filament elongation at spontaneous and gelsolin-nucleated polymerization and the turnover rates of the filament subunits at steady-state. Scallop actin nucleated nearly twice less efficient than rabbit actin. In actin filaments with free ends, when dynamics at the barbed ends overrides that at the pointed ends, the relative association rate constants of α- and β-actin were similar, whereas the relative dissociation rate constant of β-ATP-actin subunits was 2- to 3-fold higher than that of α-actin. The 2- to 3-fold faster polymerization of skeletal muscle versus scallop Ca-actin was preserved with gelsolin-capped actin filaments when only polymerization at the pointed end is possible. With gelsolin-induced polymerization, the rate constants of dissociation of ATP-actin subunits from the pointed ends were similar, while the association rate constant of β-actin to the pointed filament ends was twice lower than that of α-actin. This difference may be of physiological relevance for functional intracellular sorting of actin isoforms.

Tropomodulin Increases the Critical Concentration of Barbed End-capped Actin Filaments by Converting ADP·Pi-actin to ADP-actin at All Pointed Filament Ends

The pointed end capping protein, tropomodulin, increases the critical concentration of barbed end capped actin, i.e. it lowers the apparent affinity of pointed ends for actin monomers. We show here that this is due to the conversion of pointed end ADP. P(i)-actin (low critical concentration) to ADP-actin (high critical concentration) when 70-98% of the ends are capped by tropomodulin. We propose that this is due to the low affinity of tropomodulin for pointed ends (K(d) approximately 0.3 microM), which allows tropomodulin to rapidly exchange binding sites and transiently block access of actin monomers to all pointed ends. This leaves time for ATP hydrolysis and phosphate release to go to completion between successive monomer additions to the pointed end. When the affinity of tropomodulin for pointed ends was increased about 1000-fold by the presence of tropomyosin (K(d) < 0.05 nM), capping of 95% of the ends by tropomodulin did not alter the critical concentration. However, the critical concentration did increase when the tropomodulin concentration was raised to the high values effective in the absence of tropomyosin. This may reflect transient tropomodulin binding to tropomyosin-free actin molecules at the pointed ends of the tropomyosin-actin filaments without a high affinity tropomodulin cap, i.e. the ends that determine the value of the actin critical concentration.

Regulation of actin filament length in erythrocytes and striated muscle

Actin filaments polymerize in vitro to lengths which display an exponential distribution, yet in many highly differentiated cells they can be precisely maintained at uniform lengths in elaborate supramolecular structures. Recent results obtained using two classic model systems, the erythrocyte membrane cytoskeleton and the striated muscle sarcomere, reveal surprising similarities and instructive differences in the molecules and mechanisms responsible for determining and maintaining actin filament lengths in these two systems. Tropomodulin caps the slow-growing, pointed filament ends in muscle and in erythrocytes. CapZ caps the fast-growing, barbed filament ends in striated muscle, whereas a newly discovered barbed end capping protein, adducin, may cap the barbed filament ends in erythrocytes. The mechanisms responsible for specifying the characteristic filament lengths in these systems are more elusive and may include strict control of the relative amounts of actin filament capping proteins and side-binding proteins, molecular templates (e.g. tropomyosin and nebulin) and/or verniers (e.g. tropomyosin).

Tropomodulin caps the pointed ends of actin filaments.

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.

Coordinated inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein

Actin is an abundant intracellular protein that is released into the blood during tissue injury and its injection into rats causes microthrombi to form in the vasculature. This report and others have shown that actin filaments are able to aggregate platelets in an adenosine diphosphate (ADP)-dependent manner. The effects on this process of two plasma actin-binding proteins, vitamin D-binding protein (DBP) and gelsolin, were examined separately and together. The addition of DBP, a monomer-binding protein, to actin filaments did not affect their ability to induce platelet aggregation. However, severing of actin filaments with gelsolin resulted in an increased degree of platelet aggregation. Preincubation of F-actin with both gelsolin and DBP resulted in a significant inhibition of aggregation. The effects of DBP and gelsolin on actin-induced aggregation paralleled their effects on exchange of actin-bound adenine nucleotides. DBP inhibited 1, N6- ethenoadenosine 5′ triphosphate (epsilon-ATP) exchange with G-actin but not with F-actin. Gelsolin increased epsilon-ATP exchange with F-actin, which was largely abrogated by the addition of DBP. These results suggest that gelsolin's severing (and subsequent capping) of actin filaments not only results in an increase in the number of pointed filament ends but also in the dissociation of actin monomers containing ADP. Phalloidin, which stabilizes actin filaments while decreasing both monomer and nucleotide exchange, inhibited actin-induced aggregation, as well, indicating that depolymerization of actin filaments is not required to inhibit aggregation. Platelet activation by either G- or F- actin may thus be regulated by the local concentrations of the plasma actin-binding proteins gelsolin and DBP. Together, these proteins inhibit platelet aggregation in a manner that can be explained by their effects on actin's filament structure and the accessibility of its bound ADP. Depletion of DBP or gelsolin may allow actin released from injured tissues to stimulate purinergic receptors on platelets, and perhaps other cells, via its bound adenine nucleotides.

Coordinated Inhibition of Actin-Induced Platelet Aggregation by Plasma Gelsolin and Vitamin D-Binding Protein

AbstractActin is an abundant intracellular protein that is released into the blood during tissue injury and its injection into rats causes microthrombi to form in the vasculature. This report and others have shown that actin filaments are able to aggregate platelets in an adenosine diphosphate (ADP)- dependent manner. The effects on this process of two plasma actin-binding proteins, vitamin D-binding protein (DBP) and gelsolin, were examined separately and together. The addition of DBP, a monomer-binding protein, to actin filaments did not affect their ability to induce platelet aggregation. However, severing of actin filaments with gelsolin resulted in an increased degree of platelet aggregation. Preincubation of F-actin with both gelsolin and DBP resulted in a significant inhibition of aggregation. The effects of DBP and gelsolin on actin-induced aggregation paralleled their effects on exchange of actin-bound adenine nucleotides. DBP inhibited 1, N6-ethenoadenosine 5′ triphosphate (ϵ-ATP) exchange with G-actin but not with F-actin. Gelsolin increased ϵ-ATP exchange with F-actin, which was largely abrogated by the addition of DBP. These results suggest that gelsolin’s severing (and subsequent capping) of actin filaments not only results in an increase in the number of pointed filament ends but also in the dissociation of actin monomers containing ADP. Phalloidin, which stabilizes actin filaments while decreasing both monomer and nucleotide exchange, inhibited actin-induced aggregation, as well, indicating that depolymerization of actin filaments is not required to inhibit aggregation. Platelet activation by either G- or F-actin may thus be regulated by the local concentrations of the plasma actin-binding proteins gelsolin and DBP. Together, these proteins inhibit platelet aggregation in a manner that can be explained by their effects on actin’s filament structure and the accessibility of its bound ADP. Depletion of DBP or gelsolin may allow actin released from injured tissues to stimulate purinergic receptors on platelets, and perhaps other cells, via its bound adenine nucleotides.

Tropomyosin prevents depolymerization of actin filaments from the pointed end.

Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.

Tropomyosin stabilizes the pointed end of actin filaments by slowing depolymerization

Tropomyosin is postulated to confer stability to actin filaments in nonmuscle cells. We have found that a nonmuscle tropomyosin isolated from the intestinal epithelium can directly stabilize actin filaments by slowing depolymerization from the pointed, or slow-growing, filament end. Kinetics of elongation and depolymerization from the pointed end were measured in fluorescence assays using pyrenylactin filaments capped at the barbed end by villin. The initial pointed end depolymerization rate in the presence of tropomyosin averaged 56% of the control rate. Elongation from the pointed filament end in the presence of tropomyosin occurred at a lower free G-actin concentration, although the on rate constant, kappa p+, was not greatly affected. Furthermore, in the presence of tropomyosin, the free G-actin concentration was lower at steady state. Therefore, nonmuscle tropomyosin stabilizes the pointed filament end by lowering the off rate constant, kappa p-.

Beta-actinin: a capping protein at the pointed end of thin filaments in skeletal muscle.

We examined the function of beta-actinin as a pointed end capping protein of thin filaments in skeletal muscle. An improvement in preparing beta-actinin yielded purified beta-actinin which retained its activity for more than a week. Two-dimensional gel electrophoresis showed that the two subunits, beta I and beta II, of beta-actinin are, respectively, split into two to three components (isoforms) with different isoelectric points. Polyclonal antibody was raised by injecting such purified and undenatured chicken breast muscle beta-actinin composed of several components into a rabbit. Immuno-gold labeling examination with electron microscopy of an F-actin-beta-actinin complex decorated with HMM showed that 85% of bound gold particles was on the pointed end of actin filaments, while the remaining 15% was on the barbed end. This suggests that in beta-actinin preparation pointed end and barbed end capping proteins inevitably coexist. Immunofluorescence and immunoelectron microscopy directly showed that beta-actinin is located at the pointed end of thin filaments in myofibrils; it was also suggested that a capping protein having common antigenic determinants to beta-actinin is located at Z-line. Thus, the physiological function of beta-actinin as a pointed end capping protein was examined as follows: When beta-actinin was dissociated from the pointed end of thin filaments in an I-Z-I brush by using a high salt solution, thin filaments could be disassembled at the pointed ends at concentrations of exogenous actin lower than a critical value. At a physiological ionic strength, these salt-washed thin filaments gradually shortened at a constant rate of about 45 nm/h. Both the association and dissociation of monomeric actin at the pointed end were suppressed by the rebinding of exogenous beta-actinin. The main physiological role of beta-actinin is therefore to stabilize thin filaments in the sarcomere by preventing addition and removal of actin monomers at the pointed filament end.

Kinetics of actin elongation and depolymerization at the pointed end.

We measured the rate of elongation at the pointed filament end with increasing concentrations of G-actin [J(c) function] using villin-capped actin filaments of very small (actin/villin = 3, VA3) and relatively large size (actin/villin = 18, VA18) as nuclei for elongation. The measurements were made under physiological conditions in the presence of both Mg2+ and K+. In both cases the J(c) function was nonlinear. In contrast to the barbed filament end, however, the slope of the J(c) function sharply decreased rather than increased when the monomer concentration was lowered to concentrations near and below the critical concentration c infinity. At zero monomer concentration, depolymerization at the pointed end was very slow with a rate constant of 0.02 s-1 for VA18. When VA3 was used, the nonlinearity of the J(c) function was greatly exaggerated, and the nuclei elongated at actin concentrations below the independently measured critical concentration for the pointed end. This is consistent with and confirms our previous finding [Weber, A., Northrop, J., Bishop, M. F., Ferrone, F. A., & Mooseker, M. S. (1987) Biochemistry (preceding paper in the issue)] that at an actin-villin ratio of 3 a significant fraction of the villin is free and that a series of steady states exist between villin-actin complexes of increasing size and G-actin. The rate constant of elongation seems to increase with increasing G-actin concentrations because of increasing conversion of free villin into villin-actin oligomers during the period of the measurement of the initial elongation rate. The villin-actin oligomers have a much higher rate constant of actin binding than does free villin.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleation of actin polymerization by villin and elongation at subcritical monomer concentration.

We have obtained a quantitative description of villin-nucleated actin polymerization in physiological salt by determining the concentrations of free villin (V), villin-actin monomer (VA), villin-actin dimer (VA2), and villin-actin oligomer (VAn). Over a range of actin-villin ratios from 0.1 to 20 we determined the concentration of actin-bound villin by measuring the low-intensity pyrenylactin fluorescence of the two terminal actins in each villin-actin polymer. (To this end we first showed that each villin-actin oligomer and polymer contains two low-intensity pyrenylactin molecules.) We determined the concentration of free villin using a calibrated cutting activity assay. The pattern of increase in bound villin together with the pattern of increase in high-intensity pyrenylactin fluorescence with increasing G-actin concentration indicated, first, that villin-actin monomers were not formed at detectable levels even at a 12-fold villin excess over actin. Second, there was no stoichiometric villin-actin dimer formation at actin-villin ratios of 2. Instead there was an equilibrium between free villin, VA2, and VAn. Defining K1 = [VA]/[V][A] and K2 = [VA2]/[VA][A], a good fit of the data was obtained with K1 much less than K2 and a value of K1K2 = Kv = 10(12)-10(13) M-2 = [VA2]/[V][A]2, i.e., 1/Kv1/2 = (0.3-1) X 10(-6) M. We have assumed here that the monomer binding constant of VA2 to form VA3 was equal to the monomer binding constant of pointed filament ends, K infinity = 1/c infinity, obtained as described below.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct measurement of critical concentrations and assembly rate constants at the two ends of an actin filament

Actin filament bundles isolated from Limulus sperm were used for quantitative electron microscope studies of F-actin assembly. The assembly rate constants were calculated. In addition, the critical concentrations (C os) for both filament ends were directly determined. In 75 mM KCI and 1–5 mM Mg ++, the C os were 0.1 μM and 0.5 μM for the barbed and pointed ends, respectively. Substitution of Ca ++ (20–200 μM) for Mg ++ resulted in C os of 0.4 μM for both filament ends. Consistent with these findings, filament growth occurred only from the barbed ends of Limulus bundles “seeded” into F-actin solutions in KCI and Mg ++. Finally, filaments originally grown from the pointed filament ends of Limulus bundles were gradually lost as the actin solution reached steady state. These results demonstrate that actin filaments can “treadmill” under physiological conditions, albeit at very slow rates.

Direct electron microscopic visualization of barbed end capping and filament cutting by intestinal microvillar 95-kdalton protein (villin): a new actin assembly assay using the Limulus acrosomal process.

We have re-examined the Ca(++)-dependent interaction of an intestinal microvillar 95- kdalton protein (MV-95K) and actin using the isolated acrosomal process bundles from limulus sperm. Making use of the processes as nuclei for assembling actin filaments, we quantitatively and qualitatively examined MV-95K's effect on filament assembly and on F- actin, both in the presence and in the absence of Ca(++). The acrosomal processes are particularly advantageous for this approach because they nucleate large numbers of filaments, they are extremely stable, and their morphology can be used to determine the polarity of any nucleated filaments. When filament nucleation was initiated in the presence of MV-95K and the absence of Ca(++), there was biased filament assembly from the bundle ends. The calculated elongation rates from both the barbed and pointed filament ends were virtually indistinguishable from control preparations. In the presence of Ca(++), MV-95K completely inhibited filament assembly from the barbed filament end without affecting the initial rate of assembly from the pointed filament end. The inhibition of assembly results from MV-95K binding to and capping the barbed filament end, thereby preventing monomer addition. This indicates that, while MV-95K is a potent nucleator of actin assembly, it is also a potent inhibitor of actin filament elongation. To examine the effects of MV-95K on F-actin in the presence of Ca(++), we developed an assay where MV-95K is added to filaments previously assembled from acrosomal processes without causing filament breakage during mixing. These results clearly demonstrated that rapid filament shortening by MV-95K results through a mechanism of disrupting intrafilament monomer-monomer interactions. Finally, we show that tropomyosin-containing actin filaments are insensitive to cutting, but not to capping, by MV-95K in the presence of Ca(++).