FCHSD1 and FCHSD2 Are Expressed in Hair Cell Stereocilia and Cuticular Plate and Regulate Actin Polymerization In Vitro
Figure 5
FCHSD1, but not FCHSD2, binds to SNX9 and enhances SNX9’s activity in stimulating WASP-Arp2/3-mediated F-actin polymerization in vitro.
(A) Western blots showing that FCHSD1, but not FCHSD2, was co-immunoprecipitated with SNX9. (B) Schematic drawing of the domain structures of FCHSD1 and SNX9 that are used in the co-immunoprecipitation experiments in (C) and (D). (C) Western blots showing that the F-BAR domain of FCHSD1 was co-immunoprecipitated with SNX9. (D) Western blots showing that the SH3 domain of SNX9 was co-immunoprecipitated with FCHSD1. Expression vectors were transfected into HEK293 cells to express epitope-tagged proteins, and cell lysates were subjected to immunoprecipitation. IP indicates antibody used for immunoprecipitation and WB indicates antibody used for detection. The immunoglobulin heavy and light chains are indicated as “H” and “L”, respectively. (E) GST-SNX9 stimulated WASP-Arp2/3-mediated F-actin polymerization in vitro, which was enhanced by GST-FCHSD1ΔC. (F) Both GST-SNX9 and GST-FCHSD2ΔC stimulated WASP-Arp2/3-mediated F-actin polymerization in vitro, but there was no synergistic effect between these two proteins. Monomeric actin (3 µM, 10% pyrene-labeled) was incubated with 1 µM GST-FCHSD1ΔC (or GST-FCHSD2ΔC, or GST-SNX9) in the presence of 40 nM His-WASP145 and 20 nM Arp2/3 protein complex, and F-actin polymerization was tracked by monitoring the increase of in pyrene fluorescence using a spectrofluorometer. a.u., absorbance units.