100x oil immersion objective na1.49  (Nikon)

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2025.108550

Figure Lengend Snippet: 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.

Article Snippet: TIRF microscopy was performed using a Nikon Eclipse Ti2 inverted microscope equipped with an Apo TIRF 100x oil immersion objective, two solid-state lasers, and a cooled, back-illuminated ORCA-Flash4.0 Hamamatsu digital camera.

Techniques: Microscopy, Time-lapse Microscopy, Standard Deviation, Fluorescence

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2025.108550

Figure Lengend Snippet: 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 FMN1 is inhibited via interaction with WW1-WW2 FNBP4. In Model 1, the first panel shows that the dimeric FH2 domain causes bundling of actin filaments by interacting with the sides of the filaments while binding to other filaments via its outside surface. In contrast, the first panel of Model 2 shows that the FH2 domain causes actin filament bundling by binding to filaments in a similar manner to how it binds to the barbed ends, while interacting with other filaments via its outside surface. The second panel of both Models 1 and 2 shows that interaction with WW1-WW2 FNBP4 inhibits the bundling activity of FH1-FH2 FMN1.

Article Snippet: TIRF microscopy was performed using a Nikon Eclipse Ti2 inverted microscope equipped with an Apo TIRF 100x oil immersion objective, two solid-state lasers, and a cooled, back-illuminated ORCA-Flash4.0 Hamamatsu digital camera.

Techniques: Centrifugation, Staining, SDS Page, Two Tailed Test, Microscopy, Incubation, Activity Assay, Binding Assay