Figure 6. RHGF-1 localizes along F-actin... | Rockefeller University Press

Figure 6.

$RHGF-1 localizes along F-actin in basal stress fibers and binds F-actin in vitro. (A) Maximum intensity projection of live super-resolution confocal images of RHGF-1::tagRFP and GFP::ACT-1 in an occupied spermatheca. Region in yellow box enlarged on right. Scale bars: whole spermatheca, 10 µm; enlargement, 2 µm. (B) X-Z view at the surface of the spermatheca. Scale bar, 2 µm. (C) Intensity line profile based on 5-pixel line along X-Z view in B. (D) Representative two-photon images of fluorescence intensity and pseudo-colored FLIM of GFP::ACT-1 alone and co-expressed with cytoplasmic tagRFP or RHGF-1::tagRFP, which acts as acceptor. Scale bar, 20 µm. (E) Comparison of the fluorescence lifetime of GFP::ACT-1 alone and GFP::ACT-1 with cytoplasmic tagRFP or RHGF-1::tagRFP. N ≥ 16. Error bars are mean ± SEM. Statistical comparisons were performed by Brown-Forsythe ANOVA-Dunnett’s T3 multiple comparisons test. Stars designate statistical significance (**** P < 0.0001, ns P > 0.05). N, number of samples analyzed. (F–H) High-speed sedimentation assays demonstrate direct F-actin binding of the N-terminal half of RHGF-1 (RHGF-1-N), weakened by loss of the PDZ domain. (F) In control experiments 10 µM G-actin and 5 µM of either RHGF-1-N or RHGF-1-N-ΔPDZ were incubated alone in polymerization buffer, and the proteins in the pellet (P) and supernatant (S) fractions recovered after centrifugation at 200,000 × g were stained with Coomassie blue to assess spontaneous precipitation of the RHGF-1 fragments in the absence of actin. (G) Co-sedimentation of RHGF-1-N and RHGF-1-N-ΔPDZ with F-actin in high-speed pelleting assays. Increasing concentrations of RHGF-1-N and RHGF-1-N-ΔPDZ as indicated were incubated with 10 µM G-actin in polymerization buffer, and the proteins recovered in the pellet (P) and the supernatant (S) fractions after the centrifugation at 200,000 × g were analyzed as above. (H) Quantification of equilibrium constants of RHGF-1-N and RHGF-1-N-ΔPDZ with F-actin from experiments as shown in G. Solid pink and gray lines represent calculated binding isotherms. Shown values depict calculated KD values and respective standard errors of the fit. Source data are available for this figure: SourceData F6.$

RHGF-1 localizes along F-actin in basal stress fibers and binds F-actin in vitro. (A) Maximum intensity projection of live super-resolution confocal images of RHGF-1::tagRFP and GFP::ACT-1 in an occupied spermatheca. Region in yellow box enlarged on right. Scale bars: whole spermatheca, 10 µm; enlargement, 2 µm. (B) X-Z view at the surface of the spermatheca. Scale bar, 2 µm. (C) Intensity line profile based on 5-pixel line along X-Z view in B. (D) Representative two-photon images of fluorescence intensity and pseudo-colored FLIM of GFP::ACT-1 alone and co-expressed with cytoplasmic tagRFP or RHGF-1::tagRFP, which acts as acceptor. Scale bar, 20 µm. (E) Comparison of the fluorescence lifetime of GFP::ACT-1 alone and GFP::ACT-1 with cytoplasmic tagRFP or RHGF-1::tagRFP. N ≥ 16. Error bars are mean ± SEM. Statistical comparisons were performed by Brown-Forsythe ANOVA-Dunnett’s T3 multiple comparisons test. Stars designate statistical significance (**** P < 0.0001, ns P > 0.05). N, number of samples analyzed. (F–H) High-speed sedimentation assays demonstrate direct F-actin binding of the N-terminal half of RHGF-1 (RHGF-1-N), weakened by loss of the PDZ domain. (F) In control experiments 10 µM G-actin and 5 µM of either RHGF-1-N or RHGF-1-N-ΔPDZ were incubated alone in polymerization buffer, and the proteins in the pellet (P) and supernatant (S) fractions recovered after centrifugation at 200,000 × g were stained with Coomassie blue to assess spontaneous precipitation of the RHGF-1 fragments in the absence of actin. (G) Co-sedimentation of RHGF-1-N and RHGF-1-N-ΔPDZ with F-actin in high-speed pelleting assays. Increasing concentrations of RHGF-1-N and RHGF-1-N-ΔPDZ as indicated were incubated with 10 µM G-actin in polymerization buffer, and the proteins recovered in the pellet (P) and the supernatant (S) fractions after the centrifugation at 200,000 × g were analyzed as above. (H) Quantification of equilibrium constants of RHGF-1-N and RHGF-1-N-ΔPDZ with F-actin from experiments as shown in G. Solid pink and gray lines represent calculated binding isotherms. Shown values depict calculated K_D values and respective standard errors of the fit. Source data are available for this figure: SourceData F6.