Figure 3.
TPRN bundles F-actin filaments in vitro. (A) Coomassie blue-stained SDS-PAGE gel showing purified TPRN after expression in E. coli. A TRX tag was added to the N terminus in-frame with mouse TPRN, which improves its solubility and stability. The first lane is the protein ladder. The second lane is the purified TRX-TPRN protein (arrow), the size of which is ∼100 kDa. (B–D) Actin filaments (green) in the presence of control TRX tag only (B) or TRX-TPRN (magenta) (C and D). Actin (3 μM) was incubated with 0.1 μM TRX or TRX-TPRN at RT for 1.5 h. Using an antibody against TRX, the localization of TRX-TPRN (magenta) along F-actin filamentous structures (green) was visualized by fluorescence microscopy. Note, TRX tag alone (magenta) did not bind to F-actin filaments as shown in B. (D) TRX-TPRN (magenta) was distributed along the length of F-actin filamentous structures (green) and not concentrated just at the ends of F-actin. Scale bars in B–D: 5 μm. (E) Negative stain TEM images of F-actin polymerized with 100 nM TRX, 100 nM TRX-TPRN, 300 nM TRX-TPRN, 300 nM ESPN, or 1 µM ESPN. Images acquired at a low magnification (upper panels) and high magnification (lower panels). The short green lines across the bundles show the examples of bundle diameter measurements. ESPN is used at 300 nM or above since only a few bundles appear at 100 nM. TRX-TPRN bundles show bending (black arrows), while ESPN bundles occasionally appear kinked (black arrowheads). TRX-TPRN and ESPN bundle actin filaments (red arrows) with bifurcations (open arrows). Filaments bundled by 100 nM TRX-TPRN show single filaments branched from bundles (open red arrowheads). TRX samples occasionally show filaments neighboring one another (red arrowheads), which are analyzed as pseudo-bundles in F and G. Scale bars: 200 nm (E, upper panels) and 50 nm (E, lower panels). (F) Average diameter of F-actin bundles. Compared with the diameter of control 100 nM TRX (0.022 ± 0.005 µm, average ± SD, n = 46), the bundle diameter is significantly larger for 100 nM TRX-TPRN (0.035 ± 0.018, n = 102), 300 nM TRX-TPRN (0.041 ± 0.013 µm, n = 138), 300 nM ESPN (0.071 ± 0.034 µm, n = 87), and 1 µM ESPN (0.082 ± 0.035 µm, n = 73). One-way ANOVA shows P < 0.0001. Post hoc multiple comparisons by Tukey (****P < 0.0001; ***P = 0.0001; *P < 0.05; n.s.: P ≥ 0.05). (G) Average distances between filaments increasing in the order of TRX, TRX-TPRN, and ESPN. The concentration of TRX-TPRN and ESPN does not affect the distances between filaments. One-way ANOVA shows P < 0.0001 (8.7 ± 1.9 nm, n = 52 for 100 nM TRX; 9.5 ± 2.0 nm, n = 122 for 100 nM TRX-TPRN; 8.8 ± 1.7 nm, n = 86 for 300 nM TRX-TPRN; 11.1 ± 2.4 nm, n = 134 for 300 nM ESPN; 11.5 ± 3.0 nm, n = 111 for 1 µM ESPN). Post hoc multiple comparisons by Tukey (****P < 0.0001; n.s.: P ≥ 0.05). (H) Actin-bundling by EGFP-TPRN in COS-7 cell nucleus. EGFP-TPRN (green), phalloidin (magenta), DAPI (blue). Note the forked/conjoined areas of actin bundles outlined by circles resemble the bifurcated bundles of purified TRX-TPRN in D and E. Scale bars, 10 μm. (I) A low-speed (13,500 × g) co-sedimentation assay was used to determine the cross-linking activity of TPRN. Actin (3 μM) was incubated alone or with 0.1 μM TRX-TPRN or 6 μM TRX protein or 3 μM α-actinin or 3 μM BSA under polymerization conditions, followed by centrifugation. Equivalent amounts of supernatant (S) and pellet (P) were separated using SDS-PAGE and stained with Coomassie blue. The experiments were repeated at least three times. (J) Quantification of the percentage of actin in the pellet is shown in G. Data are represented as mean ± SEM. ***P < 0.001 by unpaired two-sided t test (n = 4). (K) Coomassie blue–stained protein gel showing 3 μM actin co-sedimented with TPRN of increased concentrations (13–100 nM). Immunoblotting detected TPRN in supernatant and pellet. (L) Percentage of actin in pellet is shown in I (n = 3). 100 nM TRX-TPRN was sufficient to saturate the binding sites of 3 μM F-actin. Data are represented as mean ± SEM. TPRN in I–L is tagged with TRX at its N terminus as indicated in A. Source data are available for this figure: SourceData F3.

TPRN bundles F-actin filaments in vitro. (A) Coomassie blue-stained SDS-PAGE gel showing purified TPRN after expression in E. coli. A TRX tag was added to the N terminus in-frame with mouse TPRN, which improves its solubility and stability. The first lane is the protein ladder. The second lane is the purified TRX-TPRN protein (arrow), the size of which is ∼100 kDa. (B–D) Actin filaments (green) in the presence of control TRX tag only (B) or TRX-TPRN (magenta) (C and D). Actin (3 μM) was incubated with 0.1 μM TRX or TRX-TPRN at RT for 1.5 h. Using an antibody against TRX, the localization of TRX-TPRN (magenta) along F-actin filamentous structures (green) was visualized by fluorescence microscopy. Note, TRX tag alone (magenta) did not bind to F-actin filaments as shown in B. (D) TRX-TPRN (magenta) was distributed along the length of F-actin filamentous structures (green) and not concentrated just at the ends of F-actin. Scale bars in B–D: 5 μm. (E) Negative stain TEM images of F-actin polymerized with 100 nM TRX, 100 nM TRX-TPRN, 300 nM TRX-TPRN, 300 nM ESPN, or 1 µM ESPN. Images acquired at a low magnification (upper panels) and high magnification (lower panels). The short green lines across the bundles show the examples of bundle diameter measurements. ESPN is used at 300 nM or above since only a few bundles appear at 100 nM. TRX-TPRN bundles show bending (black arrows), while ESPN bundles occasionally appear kinked (black arrowheads). TRX-TPRN and ESPN bundle actin filaments (red arrows) with bifurcations (open arrows). Filaments bundled by 100 nM TRX-TPRN show single filaments branched from bundles (open red arrowheads). TRX samples occasionally show filaments neighboring one another (red arrowheads), which are analyzed as pseudo-bundles in F and G. Scale bars: 200 nm (E, upper panels) and 50 nm (E, lower panels). (F) Average diameter of F-actin bundles. Compared with the diameter of control 100 nM TRX (0.022 ± 0.005 µm, average ± SD, n = 46), the bundle diameter is significantly larger for 100 nM TRX-TPRN (0.035 ± 0.018, n = 102), 300 nM TRX-TPRN (0.041 ± 0.013 µm, n = 138), 300 nM ESPN (0.071 ± 0.034 µm, n = 87), and 1 µM ESPN (0.082 ± 0.035 µm, n = 73). One-way ANOVA shows P < 0.0001. Post hoc multiple comparisons by Tukey (****P < 0.0001; ***P = 0.0001; *P < 0.05; n.s.: P ≥ 0.05). (G) Average distances between filaments increasing in the order of TRX, TRX-TPRN, and ESPN. The concentration of TRX-TPRN and ESPN does not affect the distances between filaments. One-way ANOVA shows P < 0.0001 (8.7 ± 1.9 nm, n = 52 for 100 nM TRX; 9.5 ± 2.0 nm, n = 122 for 100 nM TRX-TPRN; 8.8 ± 1.7 nm, n = 86 for 300 nM TRX-TPRN; 11.1 ± 2.4 nm, n = 134 for 300 nM ESPN; 11.5 ± 3.0 nm, n = 111 for 1 µM ESPN). Post hoc multiple comparisons by Tukey (****P < 0.0001; n.s.: P ≥ 0.05). (H) Actin-bundling by EGFP-TPRN in COS-7 cell nucleus. EGFP-TPRN (green), phalloidin (magenta), DAPI (blue). Note the forked/conjoined areas of actin bundles outlined by circles resemble the bifurcated bundles of purified TRX-TPRN in D and E. Scale bars, 10 μm. (I) A low-speed (13,500 × g) co-sedimentation assay was used to determine the cross-linking activity of TPRN. Actin (3 μM) was incubated alone or with 0.1 μM TRX-TPRN or 6 μM TRX protein or 3 μM α-actinin or 3 μM BSA under polymerization conditions, followed by centrifugation. Equivalent amounts of supernatant (S) and pellet (P) were separated using SDS-PAGE and stained with Coomassie blue. The experiments were repeated at least three times. (J) Quantification of the percentage of actin in the pellet is shown in G. Data are represented as mean ± SEM. ***P < 0.001 by unpaired two-sided t test (n = 4). (K) Coomassie blue–stained protein gel showing 3 μM actin co-sedimented with TPRN of increased concentrations (13–100 nM). Immunoblotting detected TPRN in supernatant and pellet. (L) Percentage of actin in pellet is shown in I (n = 3). 100 nM TRX-TPRN was sufficient to saturate the binding sites of 3 μM F-actin. Data are represented as mean ± SEM. TPRN in I–L is tagged with TRX at its N terminus as indicated in A. Source data are available for this figure: SourceData F3.

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