Tropomyosins Regulate the Severing Activity of Gelsolin in Isoform-Dependent and Independent Manners

Figures

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  Figure 1. Binding of gelsolin to Tpm immobilized on a CMD500L sensor chip at 25°C. The Tpm isoforms were immobilized on the surface of a CMD500L sensor chip by the amine coupling method, and the reference surface was blocked with 1 M ethanolamine solution. The immobilized Tpm isoforms are (A) skTM, (B) Tpm1.12, and (C) Tpm3.1. The interactions between gelsolin and Tpm isoforms were assayed by injecting the gelsolin over the Tpm and reference surfaces at the indicated concentrations for 7 min; then, the dissociation phase was recorded by changing the gelsolin solution to polymerization buffer for 6 min. Kinetic parameters and equilibrium dissociation constants (KD) for the interactions were derived by the BIAevaluation 3.1 software. Dashed lines indicate the fits for the experimental curves assuming a 1:1 interaction model between the binding partners. The KD values in molars are indicated on the figures.

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  Figure 2. Gelsolin increases the rate of actin polymerization. (A) Gelsolin was added to 3 μM G-actin at different final concentrations and polymerization was followed by pyrene fluorescence. Data are representative curves from experiments repeated four times. (B) The same results as in (A) are plotted on a shorter timescale to demonstrate the disappearance of the initial lag phase of nucleation as an effect of gelsolin. (C) Representative actin polymerization rates calculated from the slope of the initial 5–25% segment relative to the plateau fluorescence intensity. To see this figure in color, go online.

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  Figure 3. Tropomyosins enhance the effect of gelsolin on actin polymerization. (A) In these assays, 2 μM actin was polymerized alone or in the presence of either 4.5 nM gelsolin or 4.5 nM gelsolin preincubated with different Tpm isoforms. Gelsolin and Tpms were prepared in stock solutions for 30 min after mixing them at 300 nM and 10 μM concentrations, respectively. (B) The enhancement of the polymerization rate with Tpm-complexed gelsolin over gelsolin alone was calculated from the slope of the 5–25% segment of the pyrenyl traces relative to the plateau fluorescence intensity (n = 6; data are represented as the mean ± SE). The statistical significance level for all three Tpm isoforms was p < 0.005. (C) Polymerization curves for the same amount of actin as in (A) with and without Tpms and without gelsolin. To see this figure in color, go online.

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  Figure 4. Tropomyosins enhance the actin-filament severing activity of gelsolin. For the depolymerization assays, 3 μM actin was polymerized alone or in the presence of 30 μM Tpm1.12, 30 μM Tpm3.1, or 10 μM skTM. The samples were diluted into polymerization buffer to a final concentration of 100 nM to induce spontaneous depolymerization. Gelsolin (300 nM) was prepared with or without 10 μM Tpm and was added to a 4 nM final concentration concomitant with the dilution of F-actin. Results are shown for Tpm1.12 (A), Tpm3.1 (B), and skTM (C). In these experiments, buffers for dilution were prepared from calcium-free polymerization buffer supplemented with 0.1 M CaCl2 to the desired concentration. In the left column, representative depolymerization curves of parallel measurements are plotted. In the middle column, net depolymerization rates are shown calculated from the initial nearly linear 2-min parts of the dissociation curves. (The color scheme is black, actin; red, actin/Tpm; green, actin + gelsolin; blue, actin + gelsolin/Tpm; cyan, actin/Tpm + gelsolin; magenta, actin/Tpm + gelsolin/Tpm.) In the right column, dependence of the depolymerization rate on calcium concentration is plotted for…

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  Figure 5. Tropomyosins differently protect actin filaments from depolymerization by gelsolin. (A and B) In the co-sedimentation study, 25 μM F-actin was treated with 2 μM gelsolin for 1 h, then diluted to 10 μM (gelsolin to 0.8 μM) in the presence of either 40 μM Tpm1.12, 40 μM Tpm3.1, or 10 μM skTM. The samples were incubated for 2 h, then ultracentrifuged, and the pellets were analyzed by densitometry. Controls containing only actin filaments and complexes of actin with gelsolin were also prepared. (A) Bars represent the relative sedimented actin content compared with F-actin alone (The color scheme is black, actin/gelsolin; red, actin/gelsolin + Tpm3.1; green, actin/gelsolin + Tpm1.12; blue, actin/gelsolin + skTM). (B) Bars indicate the relative gelsolin content compared with the actin/gelsolin sample (colors are the same as for A). (C and D) In the reverse experiment, first, 10 μM F-actin was prepared for 2 h alone or with 40 μM Tpm1.12, 40 μM Tpm3.1, or 10 μM skTM, and then, 0.8 μM gelsolin was added for 1 h. Bars and colors are the same as in (A) and (B). Data are given as the mean ± SE; n = 3. (E) Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the co-sedimentation…

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  Figure 6. Models of Tpm (gray and black coils) and gelsolin G1–G3 (red ribbons) binding to an actin filament (subunits represented as surfaces in pastel shades). (A) Gelsolin domains from the G1–G3/actin x-ray structure (PDB: 3FFK) (27) docked onto the cryo-EM structure of 3.5 Å mouse skTM/actin (PDB: 5JLF) (42) through superimposition of an actin protomer structure (pink). In this model, G1 does not sterically clash with Tpm (cyan circle), but G2 has significant overlap (yellow circle). (B and C) Close-ups of the Tpm/G2 interaction (yellow circle) generated from the 8 Å cryo-EM structure of chicken gizzard Tpm/actin (PDB: 3J4K) (43) (B) and from the cryo-EM structure of 3.9 Å human cytoplasmic Tpm3/actin/myosin (PDB: code 5JLH) (42) (C). These models are not intended to be accurate representations of a gelsolin/actin/Tpm interaction, but rather to indicate that the known position of Tpms on actin are in locations that could have a variety of competitive effects on gelsolin binding. Thus, the positional placement of Tpms on the surface of the actin filament will dictate whether gelsolin can simultaneously bind to the same actin filament. To see this figure in color, go online.