Nuclear protein FNBP4: A novel inhibitor of non-diaphanous formin FMN1-mediated actin cytoskeleton dynamics

Figures

• 

  Figure 1. FNBP4 is an inhibitor of FMN1. Schematic illustrations of purified constructs of FMN1 (A) and FNBP4 (B) used for invitro experiments. Purified N-terminal WW1-WW2 FNBP4, ΔWW1 FNBP4, C-ter FH1-FH2 FMN1, C-ter FH2 FMN1, and FH1 FMN1 constructs analyzed by Coomassie-stained 10% SDS-PAGE (C). Pyrene-actin polymerization assay (2 μM actin monomer, 10% pyrene labeled): Actin monomers were polymerized in the presence of 50 nM FH1-FH2 FMN1 and/or increasing concentrations of N-terminal WW1-WW2 FNBP4 (D), and (F) 50 nM FH1-FH2 FMN1 and/or increasing concentrations of N-terminal ΔWW1 FNBP4. Concentration-dependent inhibitory effects of N-terminal WW1-WW2 FNBP4 (E) and N-terminal ΔWW1 FNBP4 (G) on FH1-FH2 FMN1 under the conditions outlined in (D) and (F), respectively. Percentage of FH1-FH2 FMN1 activity was quantified by dividing the slope of the actin polymerization curve in the presence of N-terminal WW1-WW2 FNBP4 or ΔWW1 FNBP4 to the slope of the curve obtained in the absence of N-terminal WW1-WW2 FNBP4 or ΔWW1 FNBP4. Actin polymerization rates were measured from the slopes of the curves between 200 and 300 s and the IC50 value was calculated as the concentration at which the…

• 

  Figure 2. N-terminal WW1-WW2 FNBP4 inhibits FH1-FH2 FMN1-mediated actin nucleation, observed by TIRF microscopy.A, time-lapse microscopy images of actin filament assembly under the conditions specified in (i) actin only, (ii) with 50 nM FH1-FH2 FMN1, (iii) 50 nM FH2 FMN1, (iv) with 50 nM FH1-FH2 FMN1 and 400 nM N-terminal WW1-WW2 FNBP4, and (v) with 50 nM FH2 FMN1 and 400 nM N-terminal WW1-WW2 FNBP4. The scale bar represents 10 μm. B, quantification of the number of filaments at the end point was performed in four different fields. Error bars represent the standard deviation. Statistical significance was determined using a one-way ANOVA with post hoc Tukey HSD test. Significance levels are denoted as ∗∗ for p < 0.01 and ns (not significant). HSD, honestly significant difference; TIRF, total internal reflection fluorescence.

• 

  Figure 3. FH1 FMN1 attenuates the inhibitory activity of WW1-WW2 FNBP4 on FH1-FH2 FMN1-mediated actin nucleation.A, pyrene-actin polymerization assay (2 μM actin monomer, 10% pyrene labeled) containing 50 nM FH1-FH2 FMN1 in the presence or absence of 400 nM N-terminal WW1-WW2 FNBP4, and varying concentrations of FH1-FMN1. B, the plot depicts the inhibition kinetics of WW1-WW2 FNBP4 on FH1-FH2 FMN1 in the presence of FH1 FMN1, where the activity of FH1-FH2 FMN1 is plotted against the concentration of FH1 FMN1. Percentage of FH1-FH2 FMN1 activity was quantified by dividing the slope (200 and 300 s) of the actin polymerization curve for N-terminal WW1-WW2 FNBP4 and FH1-FH2 FMN1, in the presence or absence of FH1 FMN1, by the slope of the curve obtained in the FH1-FH2 FMN1 control.

• 

  Figure 4. N-ter WW1-WW2 FNBP4 inhibits FH1-FH2 FMN1 processive capping activity.A, effects of the N-terminal WW1-WW2 FNBP4 on FH1-FH2 FMN1 during the barbed-end growth of actin filaments, in the presence and absence of the capping protein (CapZ). An elongation assay was performed by adding actin monomers (0.5 μM, 10% pyrene-labeled) to mechanically sheared unlabeled actin seeds (333 nM) in the presence of 50 nM FH1-FH2 FMN1 and/or 400 nM WW1-WW2 FNBP4, with or without CapZ (200 nM). B, the plot depicts comparative analysis of the elongation rate as the slope of the fluorescence curve between 50 and 100 s of the time course, for each condition outlined in (A). Statistical significance was determined using an unpaired two-tailed Student's t test in GraphPad Prism 8. Significance levels are denoted as ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), ∗∗∗ (p ≤ 0.001), ∗∗∗∗ (p ≤ 0.0001), and ns (not significant). C, a schematic representation showing the role of WW1-WW2 FNBP4 and FH1-FH2 FMN1 interaction in actin filament elongation. In the first scenario, both CapZ and FH1-FH2 FMN1 can bind to the barbed end of actin filaments. In the second scenario, FH1-FH2 FMN1 has the ability to displace CapZ from the barbed…

• 

  Figure 5. N-terminal WW1-WW2 FNBP4 inhibits FH1-FH2 FMN1-driven actin bundling. Low-speed centrifugation assay containing preformed 5 μM F-actin and 1 μM of FH1-FH2 FMN1 (A) or 1 μM of FH2 FMN1 (C), with increasing concentrations of WW1-WW2 FNBP4. The supernatant (S) and pelleted (P) fractions were collected and analyzed by Coomassie-stained 10% SDS-PAGE. Pellets were concentrated 5-fold for better visualization. B, the plot depicts the band intensity of the actin bundle pellet from FH1-FH2 FMN1 versus WW1-WW2 FNBP4 concentrations, as shown in panel A. Statistical significance was determined using an unpaired two-tailed Student's t test in GraphPad Prism 8. Significance levels are denoted as ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), ∗∗∗ (p ≤ 0.001), ∗∗∗∗ (p ≤ 0.0001), and ns (not significant). D, TIRF microscopy images of actin bundling assay. 2 μM F-actin was incubated with FH1-FH2 FMN1 or FH2 FMN1 in the presence or absence of WW1-WW2 FNBP4 and stained with rhodamine–phalloidin. The reaction was immediately diluted 8-fold with TIRF buffer and visualized using a TIRF microscope. Scale bar is 10 μm. E, a schematic illustration depicting two possible mechanisms by which the bundling activity of FH1-FH2…

• 

  Figure 6. N-terminal WW1-WW2 FNBP4 binds to FH1 domain and the interdomain connector of FMN1.A, the best-docked pose of the full-length monomeric FMN1 and full-length FNBP4 complex. FMN1 is shown in teal blue, while FNBP4 is depicted in raspberry red. The zoomed inset highlights the binding interface, showcasing key interacting residues critical for complex formation. B, a schematic representation of the domain organization of the Extended FH2 FMN1 construct, with a zoomed-in view of the terminal FH1 and interdomain connector regions, highlighting the amino acids within a dotted red box. C, a top view of the Extended FH2 FMN1 dimer in complex with two molecules of WW1-WW2 FNBP4. Extended FH2 FMN1 dimer is shown in teal blue, while WW1-WW2 FNBP4 is shown in raspberry red. The inset displays the binding interface, highlighting the key interacting amino acids involved in the interaction. D, a plot comparing the backbone RMSD analysis between the full-length monomeric FMN1-full-length FNBP4 complex and the Extended FH2 FMN1-WW1-WW2 FNBP4 complex, calculated over the entire 10 ns MD simulation. E, binding kinetics of Extended FH2 FMN1 and WW1-WW2 FNBP4 analyzed through SPR sensorgrams. Colored…

• 

  Figure 7. N-terminal WW1-WW2 FNBP4 modulates FH2 domain activity through spatial constraints. Pyrene-actin polymerization assay (2 μM actin monomer, 10% pyrene labeled): Actin polymerization was performed using 50 nM of the Extended FH2 FMN1 construct and/or increasing concentrations of the N-terminal WW1-WW2 FNBP4 (A). The concentration-dependent inhibitory effects of N-terminal WW1-WW2 on Extended FH2 FMN1 were observed under the same conditions outlined in (A). The percentage of activity of the Extended FH2 FMN1 construct was quantified by dividing the slope (200 and 300 s) of the actin polymerization curve in the presence of N-terminal WW1-WW2 FNBP4 by the slope of the curve obtained in the absence of N-terminal WW1-WW2 FNBP4 (B). A schematic model depicting the interaction of the WW1 domain of FNBP4 with the FH1 domain and the interdomain connector between the FH1 and FH2 domains of FH1-FH2 FMN1, which poses a steric hindrance, inhibiting its actin polymerization activity (C).

• 

  Figure 8. FNBP4 contains a nuclear localization signal and localizes to the nucleus.A, immunofluorescence staining of FNBP4 in HeLa cells. Cells were fixed with a 1:1 mixture of ice-cold acetone and methanol. Nuclei were stained with DAPI, while FNBP4 was visualized using an anti-FNBP4 antibody. The images represent a single Z-stack section (middle slice). The scale bar represents 20 μm. B, the table show the amino acid sequences of FNBP4 with nuclear localization signal using cNLS mapper. KGIKRKATEI (787–797 aa) and RARLKRRKMA (1005–1015 aa) are predicted as monopartite nuclear localization signals. C, schematic representations of GFP-tagged various FNBP4 (full or truncated) constructs utilized for transient overexpression in HeLa cells. KGIKRKATEI (787–797 aa) and RARLKRRKMA (1005–1015 aa) represented as NLS1 and NLS2, respectively. D, representative images show subcellular distributions of GFP-tagged different truncated FNBP4 constructs in HeLa cells. HeLa cells were transiently transfected with either the vector alone or GFP-tagged FNBP4 (full or truncated) constructs, followed by fixation with PFA and DAPI staining of the nuclei. The right-side plot shows the fluorescence intensity…