Figure 4. IRSp53 can bind to and bundle... | Rockefeller University Press

$IRSp53 can bind to and bundle actin filaments. (A) Representative images showing PSD condensation on actin network when 5× PSD, each component at 0.5 μM, was mixed with 1 μM of G-actin and incubated for 20 min at room temperature. 2% of PSD-95, IRSp53, and Shank3 were labeled with different fluorophores as indicated. 10% of actin was labeled with Rhodamine dye. GKAP and Homer3 were not labeled and are thus invisible. Images were collected as a z series maximum projection. (B) Zoom-in view of PSD condensates with Rhd-actin. (C) FRAP analysis showing the fluorescence recovery of actin and PSD-95 following a bleaching event. The recovery of fluorescence signals was quantified and plotted. Data are obtained from three condensate/actin regions and are presented as mean ± SD. (D) IRSp53 can bundle actin filaments in aqueous solution. 1 μM of actin was incubated with increasing amounts of IRSp53 at indicated concentrations. 10% of actin was labeled with Rhodamine dye. (E) Experimental setup to monitor possible actin binding and bundling activity of IRSp53 via low-speed (10,000 g) and high-speed (100,000 g) centrifugations. (F) Schematic diagrams of various fragments used to map the actin binding interface in IRSp53. (G) SDS-PAGE showing the level of actin bundling induced by different fragments of IRSp53 as designated in F. 1 μM actin was mixed with 4 μM of various IRSp53 constructs. The amount of actin in the pellet fraction, which represents the bundled actin filaments, is quantified from three independent batches of experiments. Data are presented as mean ± SD. ****, P < 0.0001 using one-way ANOVA with Dunnett’s multiple comparison test. (H) SDS-PAGE showing binding of various IRSp53 fragments, as designated in F, to F-actin. 1 μM actin was mixed with 4 μM of various IRSp53 constructs. Note that the pellet, obtained after ultracentrifugation, was resuspended in one quarter of the initial mixture volume for concentration. The portion of protein in the pellet fraction, which represents the actin-bound protein, is quantified from three independent batches of experiments. The amount of protein sedimented without the presence of actin filaments was subtracted for correction (Fig. S4 E). The corrected pellet ratio of protein was further divided by the pellet ratio of F-actin for normalization. Data are presented as mean ± SD. ***, P < 0.001; ****, P < 0.0001 using one-way ANOVA with Dunnett’s multiple comparison test. (I) Surface charge potential map of the IRSp53 SH3 domain (PDB accession no. 3RNJ) showing a cluster of positively charged residues (highlighted with a circle in dashed lines and enlarged to show the side chain orientations of these residues). Highlighted residues were mutated to Glu, and the mutant was referred to as IRSp53_RKE. (J) ITC-based experiment measured the binding between Shank3 and IRSp53_RKE, showing that the mutation did not affect IRSp53, via its SH3 domain, to bind to Shank3. 200 μM of Shank3 was titrated into 20 μM of IRSp53 with 200 mM NaCl in the binding buffer. (K) Representative fluorescence images showing that WT IRSp53, but not the actin binding deficient mutants (K4E, RKE), can bundle actin filaments. IRSp53 1–475 could not bundle actin filaments, and the further inclusion of 24 residues into the C-terminus (aa 1–499) rescued the actin-bundling ability to that of the full-length protein. Identical imaging settings were used for all groups. Source data are available for this figure: SourceData F4.$

Figure 4.

IRSp53 can bind to and bundle actin filaments. (A) Representative images showing PSD condensation on actin network when 5× PSD, each component at 0.5 μM, was mixed with 1 μM of G-actin and incubated for 20 min at room temperature. 2% of PSD-95, IRSp53, and Shank3 were labeled with different fluorophores as indicated. 10% of actin was labeled with Rhodamine dye. GKAP and Homer3 were not labeled and are thus invisible. Images were collected as a z series maximum projection. (B) Zoom-in view of PSD condensates with Rhd-actin. (C) FRAP analysis showing the fluorescence recovery of actin and PSD-95 following a bleaching event. The recovery of fluorescence signals was quantified and plotted. Data are obtained from three condensate/actin regions and are presented as mean ± SD. (D) IRSp53 can bundle actin filaments in aqueous solution. 1 μM of actin was incubated with increasing amounts of IRSp53 at indicated concentrations. 10% of actin was labeled with Rhodamine dye. (E) Experimental setup to monitor possible actin binding and bundling activity of IRSp53 via low-speed (10,000 g) and high-speed (100,000 g) centrifugations. (F) Schematic diagrams of various fragments used to map the actin binding interface in IRSp53. (G) SDS-PAGE showing the level of actin bundling induced by different fragments of IRSp53 as designated in F. 1 μM actin was mixed with 4 μM of various IRSp53 constructs. The amount of actin in the pellet fraction, which represents the bundled actin filaments, is quantified from three independent batches of experiments. Data are presented as mean ± SD. ****, P < 0.0001 using one-way ANOVA with Dunnett’s multiple comparison test. (H) SDS-PAGE showing binding of various IRSp53 fragments, as designated in F, to F-actin. 1 μM actin was mixed with 4 μM of various IRSp53 constructs. Note that the pellet, obtained after ultracentrifugation, was resuspended in one quarter of the initial mixture volume for concentration. The portion of protein in the pellet fraction, which represents the actin-bound protein, is quantified from three independent batches of experiments. The amount of protein sedimented without the presence of actin filaments was subtracted for correction (Fig. S4 E). The corrected pellet ratio of protein was further divided by the pellet ratio of F-actin for normalization. Data are presented as mean ± SD. ***, P < 0.001; ****, P < 0.0001 using one-way ANOVA with Dunnett’s multiple comparison test. (I) Surface charge potential map of the IRSp53 SH3 domain (PDB accession no. 3RNJ) showing a cluster of positively charged residues (highlighted with a circle in dashed lines and enlarged to show the side chain orientations of these residues). Highlighted residues were mutated to Glu, and the mutant was referred to as IRSp53_RKE. (J) ITC-based experiment measured the binding between Shank3 and IRSp53_RKE, showing that the mutation did not affect IRSp53, via its SH3 domain, to bind to Shank3. 200 μM of Shank3 was titrated into 20 μM of IRSp53 with 200 mM NaCl in the binding buffer. (K) Representative fluorescence images showing that WT IRSp53, but not the actin binding deficient mutants (K4E, RKE), can bundle actin filaments. IRSp53 1–475 could not bundle actin filaments, and the further inclusion of 24 residues into the C-terminus (aa 1–499) rescued the actin-bundling ability to that of the full-length protein. Identical imaging settings were used for all groups. Source data are available for this figure: SourceData F4.

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